US8877473B2 - Formulations of recombinant furin - Google Patents

Formulations of recombinant furin Download PDF

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US8877473B2
US8877473B2 US13/488,324 US201213488324A US8877473B2 US 8877473 B2 US8877473 B2 US 8877473B2 US 201213488324 A US201213488324 A US 201213488324A US 8877473 B2 US8877473 B2 US 8877473B2
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furin
rfurin
composition
sugar
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US20120329128A1 (en
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Meinhard Hasslacher
Andrzej Citkowicz
Catherine White
Ken Franke
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Takeda Pharmaceutical Co Ltd
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Baxter Healthcare SA
Baxter International Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21075—Furin (3.4.21.75)

Definitions

  • This application generally relates to the field of protein formulations.
  • SPC proteolytic mammalian subtilisin-like proprotein convertases
  • Furin also termed PACE (paired basic amino acid cleavage enzyme) is ubiquitously expressed in all mammalian tissues and cell lines and is capable of processing a wide range of bioactive precursor proteins in the secretory pathway, including also hormones, growth factors, receptors, viral and bacterial proteins, and plasma proteins. It is a calcium-dependent serine endoprotease structurally arranged into several domains, namely a signal peptide, propeptide, catalytic domain, middle domain, (also termed homo-B or P-domain), the C-terminally located cysteine-rich domain, transmembrane domain and the cytoplasmic tail.
  • the furin protease cleavage site comprises a recognition sequence which is characterized by the amino acid sequence Arg-X-Lys/Arg-Arg (Hosaka et al., J Biol Chem. 1991; 266:12127-30).
  • Furin belongs to the family of the pro-protein convertases and is dependent on calcium (Ca 2+ ). Furin specifically cleaves the C-terminal peptide bond of arginine within a specific sequence, containing arginine at positions ⁇ 1 and ⁇ 4. This sequence can be found in numerous human proteins, showing that furin plays a major role in the maturation of a number of human pro-proteins. Accordingly, furin is a proprotein convertase that processes latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that cleaves precursor proteins at their paired basic amino acid processing sites. One substrate for furin is von Willebrand Factor (vWF).
  • vWF von Willebrand Factor
  • Furin e.g., rfurin
  • rfurin is capable of converting pro-VWF (pro-von Willebrand factor) into mature VWF by cleaving the Arg741-Ser742 peptide bond of VWF.
  • This maturation step is part of, for example, a rVWF production process which provides a therapy for von Willebrand Disease Type B.
  • the production of activated recombinant proteins is of high clinical and diagnostic importance.
  • active or mature proteins like mature VWF, may be used to control blood coagulation.
  • Furin formulations particularly recombinant furin (rfurin) formulations, often have low shelf life (less than 6 months) due to a loss of rfurin activity from protein degradation (often from self-cleavage of the furin protein).
  • rfurin formulations when used in certain production processes, such as production of mature vWF, rfurin formulations are diluted, often by 200-fold, and the diluted rfurin generally shows a disproportionate loss of activity. Accordingly, there is a need in the field for methods and formulations that stabilize furin (e.g., rfurin) compositions, as well as methods for diluting concentrated furin (e.g., rfurin) compositions without losing a substantial fraction of furin activity.
  • the present disclosure fulfills these and other needs in the field of furin (e.g., rfurin) formulation, storage, and manipulation, by providing methods and formulations for stabilizing furin (e.g., rfurin compositions).
  • furin e.g., rfurin
  • These needs are met, at least in part, by the discovery that the addition of sugars, sugar alcohols, and/or non-ionic surfactants significantly increases the stability of furin (e.g., rfurin) compositions during storage and mechanical stress.
  • the present invention provides highly stabilized furin formulations, particularly highly stabilized rfurin formulations that show increased stability under conditions of high temperature, agitation, and varying pH as compared to the starting (control) furin formulations.
  • the present disclosure provides a stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: from 8,000 U/mL to 500,000 U/mL rfurin; from 100 mM to 300 mM of a pharmaceutically acceptable salt; from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol; from 10 to 200 ppm non-ionic surfactant; from 10 to 200 mM buffering agent; and a pH from 5.5 to 7.5.
  • rfurin furin
  • compositions provided above include 150 mM to 250 mM of a pharmaceutically acceptable salt. In another embodiment, the compositions provided above include 190 ⁇ 10 mM of a pharmaceutically acceptable salt. In another embodiment, the compositions provided above include 190 mM of a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt is sodium chloride. In another embodiment of the compositions provided above, the pharmaceutically acceptable salt is potassium chloride.
  • compositions provided above include 0.9 ⁇ 0.2 mM calcium. In another embodiment, the compositions provided above include 0.92 mM calcium.
  • compositions provided above include from 5% to 15% sugar or sugar alcohol. In another embodiment, the compositions provided above include 10 ⁇ 2% sugar or sugar alcohol. In another embodiment, the compositions provided above include 10% sugar or sugar alcohol.
  • the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof. In another embodiment of the compositions provided above, the sugar or sugar alcohol is trehalose. In another embodiment of the compositions provided above, the sugar or sugar alcohol is sucrose.
  • the compositions provided above include from 10 ppm to 100 ppm non-ionic surfactant. In another embodiment, the compositions provided above include from 50 ppm to 100 ppm non-ionic surfactant. In another embodiment, the compositions provided above include 75 ppm non-ionic surfactant.
  • the non-ionic surfactant is polysorbate-80.
  • compositions provided above include from 50 mM to 150 mM buffering agent. In another embodiment, the compositions provided above include 90 ⁇ 10 mM buffering agent. In another embodiment, the compositions provided above include 91 mM buffering agent.
  • the buffering agent comprises acetate. In another embodiment of the compositions provided above, the buffering agent comprises HEPES. In another embodiment of the compositions provided above, the buffering agent comprises MES. In another embodiment of the compositions provided above, the buffering agent comprises acetate and HEPES.
  • the compositions provided above include from 25 mM to 75 mM acetate and from 25 to 75 mM HEPES. In another embodiment, the compositions provided above include 45 ⁇ 5 mM acetate and 45 ⁇ 5 mM HEPES. In another embodiment, the compositions provided above include 45 mM acetate and 46 mM HEPES.
  • the compositions provided above have a pH from 5.5 to 7.0. In another embodiment, the compositions provided above have a pH from 5.5 to 6.5. In another embodiment, the compositions provided above have a pH of 6.0 ⁇ 0.2. In another embodiment, the compositions provided above have a pH of 6.0.
  • the composition has increased stability when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.
  • the composition maintains a higher percentage of rfurin activity when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.
  • the composition maintains a higher percentage of rfurin monomer content when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.
  • the composition has increased stability when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.
  • the composition maintains a higher percentage of rfurin activity when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.
  • the composition maintains a higher percentage of rfurin monomer content when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.
  • the composition has increased stability when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.
  • the composition maintains a higher percentage of rfurin activity when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.
  • the composition maintains a higher percentage of rfurin monomer content when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.
  • the present disclosure provides a stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: from 8,000 U/mL to 500,000 U/mL rfurin; 190 mM sodium chloride; 0.92 mM calcium; 10% trehalose; 75 ppm polysorbate 80; 45 mM acetic acid; 46 mM HEPES; and a pH from 5.5 to 7.5.
  • rfurin recombinant furin
  • the compositions provided above have a pH from 5.5 to 7.0. In another embodiment, the compositions provided above have a pH from 5.5 to 6.5. In another embodiment, the compositions provided above have a pH of 6.0 ⁇ 0.2. In another embodiment, the compositions provided above have a pH of 6.0.
  • the present disclosure provides a method for diluting an aqueous rfurin composition, the method comprising adding a dilution buffer comprising from 10 ppm to 200 ppm of a non-ionic surfactant to the aqueous rfurin composition, thereby forming a diluted rfurin composition.
  • the dilution buffer comprises from 10 ppm to 100 ppm of a non-ionic surfactant. In another embodiment of the methods provided above, the dilution buffer comprises from 50 ppm to 100 ppm of a non-ionic surfactant. In another embodiment of the methods provided above, the dilution buffer comprises 75 ppm of a non-ionic surfactant.
  • the dilution buffer has a pH of from 5.5 to 7.5. In another embodiment of the methods provided above, the dilution buffer has a pH of from 5.5 to 6.5. In another embodiment of the methods provided above, the dilution buffer has a pH of 6.0 ⁇ 0.2. In another embodiment of the methods provided above, the dilution buffer has a pH of 6.0.
  • the diluted rfurin composition comprises at least 50% of rfurin activity present in the aqueous rfurin composition prior to dilution.
  • the diluted rfurin composition comprises at least 75% of rfurin activity present in the aqueous rfurin composition prior to dilution.
  • the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin), said formulation comprising: 8,000-52,000 U/mL rfurin; 1 mM to 300 mM of a pharmaceutically acceptable salt; 0.1 mM to 2 mM calcium chloride; 1% to 20% sugar and/or sugar dihydrate; 50 to 100 ppm of a surfactant; and 1 mM to 100 mM of a carboxylic acid, a buffering agent in sufficient concentration for maintaining a pH of 4.0-7.0, or both said carboxylic acid and said buffering agent.
  • rfurin recombinant furin
  • the formulations provided above include 180-200 mM salt.
  • the salt is sodium chloride.
  • the formulations provided above include 0.5-1.0 mM calcium.
  • the formulations provided above include 5-15% sugar or sugar dihydrate.
  • the sugar or sugar dihydrate is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof.
  • the surfactant is polysorbate 80.
  • the surfactant is present at a concentration of about 65 to about 85 ppm.
  • the carboxylic acid is acetic acid.
  • the carboxylic acid is in a concentration of 35-55 mM.
  • the buffering agent is HEPES.
  • the buffering agent is in a concentration of about 20 to about 100 mM.
  • the formulation shows improved stability after storage at 37° C. as compared to a formulation that does not contain a sugar or sugar dihydrate.
  • the formulation is prepared by adding rfurin to a composition comprising 1% polysorbate 80, 500 mM HEPES, 400 mM acetic acid, 1 mM calcium chloride, and trehalose dihydrate powder, wherein said composition has a pH of 6.0 prior to addition to said rfurin.
  • the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin), said formulation comprising: 8,000-57,000 U/mL rfurin; 190 mM sodium chloride; 0.92 mM calcium chloride; 10% w/w trehalose dihydrate; 75 ppm polysorbate 80; 45 mM acetic acid; and 46 mM HEPES.
  • the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin) that includes: (a) 8,000-52,000 U/mL rfurin; (b) 1 mM to 300 mM of a pharmaceutically acceptable salt; (c) 0.1 mM to 2 mM calcium chloride; (d) 1% to 20% sugar and/or sugar dihydrate; (e) 50 to 100 ppm of a surfactant; and (f) 1 mM to 100 mM of a carboxylic acid or a buffering agent in sufficient concentration for maintaining a pH of 4.0-7.0 or both the carboxylic acid and the buffering agent.
  • FIG. 2 rFurin in the control formulation—a temperature study analyzed using SEC. The same samples as in FIG. 1 were analyzed using SEC. Relative rfurin peak heights are plotted against the number of days of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.
  • FIG. 3 rFurin in the control formulation incubated at 37° C. analyzed using SEC. rFurin samples in the control formulation were incubated at 37° C. for seven days.
  • FIG. 3 shows an overlay of SEC absorption profiles at 280 nm of six time points. The same study as in FIG. 1 .
  • FIG. 6 rFurin in the control formulation—a freeze/thaw study analyzed using SEC. rFurin peak heights are plotted against the number of freeze/thaw cycles.
  • FIG. 8 rFurin in the control formulation—an agitation study analyzed using SEC. rFurin peak heights are plotted against the time of agitation.
  • FIG. 9 Comparison of different lots of rfurin using Western Blotting. Various lots of rfurin were analyzed using Western Blotting. Production order is denoted alphabetically. Lane: (1) MWM marker; (2) furin reference; (3) 03-04-09 (lot 018342, 2 mL vial); (4) 03-08-09 (lot 018365, 2 mL vial); (5) 03-15-09 (lot 018366, 2 mL vial); (6) 03-01-09 (lot 018315, 2 mL vial); (7) 07-26-10 (lot X572, 200 mL bottle); (8) 03-06-09 (lot 018365, 200 mL bottle); (9) 08-01-10 (lot X576, 200 mL bottle); (10) 03-09-09 (lot 018366, 200 mL bottle); (11) MWM marker; (12) furin reference; (13) 08-03-10 (lot X577, 200 mL bottle); (14) 07-24-10 (lot X573, 200 mL bottle); (15)
  • FIG. 12 Effect of pH 5 and 5.5 on rfurin activity analyzed using SEC. rFurin samples were incubated at 37° C. at either pH: 5.0, 5.5 or 6.0 (control formulation). Relative peak heights of rfurin are plotted against the number of days of incubation. The relative peak heights were calculated as the percentage of the rfurin peak height at time zero.
  • FIG. 13 rFurin stability at 37° C. in the control formulation analyzed using SEC.
  • a rfurin sample in the control formulation was incubated for four days at 37° C.
  • FIG. 14 rFurin stability in MES pH 6.0 analyzed using SEC.
  • FIG. 15 rFurin stability in HEPES pH 7.0 analyzed using SEC.
  • FIG. 16 rFurin stability in HEPES pH 8.0 analyzed using SEC.
  • FIG. 17 Effects of sucrose and polysorbate 80 additives on rfurin stability at 37° C. analyzed using SEC. The samples were incubated at 37° C. for four days. Relative peak heights of rfurin are plotted against the time of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.
  • FIG. 18 Effects of sucrose and polysorbate 80 on rfurin stability in the control formulation at 37° C. analyzed using the furin activity assay.
  • the relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, control formulation plus 10% sucrose, and control formulation plus 10% sucrose plus 25 ppm polysorbate 80.
  • FIG. 19 Effects of sucrose and polysorbate 80 on rfurin stability in MES buffer at 37° C. analyzed using the furin activity assay.
  • FIG. 20 Comparison of rfurin stability at 37° C. in the control formulation to the formulation in MES buffer analyzed using the furin activity assay.
  • FIG. 22 rFurin Stability at 37° C.: sucrose vs. mannitol analyzed using SEC. The same samples as in FIG. 21 were analyzed using SEC. Relative rfurin peak heights are plotted against the time of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.
  • FIG. 23 rFurin stability in trehalose after 5 days of incubation at 37° C. analyzed by the furin activity assay.
  • the effects of varied amounts of trehalose and polysorbate 80 on rfurin stability at 37° C. are examined using the furin activity assay.
  • All samples were spiked with HEPES and acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0) then, either 10% sucrose or various amounts of trehalose and/or polysorbate 80 were added.
  • a rfurin sample in the control formulation is also included.
  • the data are sorted from the highest to lowest activity values after 5 days of incubation at 37° C. (right bar in each pair).
  • FIG. 25 rFurin stability in trehalose after 5 days of incubation at 37° C. analyzed using SEC. The same samples as in FIG. 24 . The graph shows what percentage of each sample's initial rfurin peak remained after five days of incubation at 37° C. A rfurin sample in the control formulation is also included.
  • FIG. 26 rFurin stability in trehalose after 5 days of incubation at 35° C. analyzed using Western Blotting. The same samples as in FIG. 24 . The samples are paired: first sample—time zero, second sample—incubated at 37° C. for 5 days.
  • FIG. 28 Effect of the trehalose content on rfurin stability at 37° C. analyzed using SEC after 5 days of incubation. The same experiment as in FIG. 27 . rFurin peak heights after five days of incubation at 37° C. are plotted against the percentage of trehalose added to the samples.
  • FIG. 30 Effect of Polysorbate 80 content on rfurin stability at 37° C. analyzed using SEC after 5 days of incubation. The same samples as in FIG. 29 . rFurin peak heights after five days of incubation at 37° C. are plotted against the amount of polysorbate 80 added to the samples.
  • FIG. 31 rFurin in the highly stabilized formulation—agitation study analyzed using the furin activity assay.
  • the rfurin samples in the highly stabilized formulation were spiked with various amount of polysorbate 80.
  • FIG. 32 rFurin in the highly stabilized formulation—agitation study analyzed using SEC. The same experiment as in FIG. 31 .
  • the graph shows rfurin peak heights before and after three hours of agitation. The data are sorted from the highest to the lowest. Left bars in each pair show activity at time 0, right bars in each pair show activity after agitation.
  • FIG. 34 rFurin in the highly stabilized formulation—effect of polysorbate 80 in agitation study analyzed using SEC. The same samples as in FIG. 32 . rFurin peak heights after three hours of agitation are plotted against the content of polysorbate 80.
  • FIG. 35 rFurin in the control formulation—agitation study analyzed using SEC.
  • the chromatogram shows an overlay of SEC absorption profiles at 280 nm comparing rfurin samples in the control formulation before and after three hours of agitation.
  • the rfurin peak of the agitated sample is significantly smaller than the control sample.
  • the absence of high molecular mass peaks in the agitated sample is observed (see paragraph 3.5.1 for explanation).
  • FIG. 36 UV spectra of rfurin in the control formulation (the same samples as in FIG. 35 ).
  • the UV spectrum of the rfurin sample after three hours of agitation shows a slanted and extremely elevated profile compared to the sample before agitation, which indicates the presence of a significant level of aggregates in the agitated sample.
  • FIG. 37 UV spectra of the rfurin samples in the highly stabilized formulation without polysorbate 80.
  • the UV spectrum of the rfurin sample after three hours of agitation shows an elevated profile compared to the sample before agitation, which indicates the presence of significant level of aggregates in the agitated sample.
  • FIG. 38 rFurin in the highly stabilized Formulation without polysorbate 80—an agitation study analyzed using SEC.
  • the chromatogram shows an overlay of SEC absorption profiles at 280 nm comparing rfurin samples in the highly stabilized formulation without polysorbate 80 before and after three hours of agitation.
  • the rfurin peak of the agitated sample is significantly smaller than the control sample. The absence of high molecular mass peaks is observed.
  • FIG. 39 UV spectra of the rfurin samples in the highly stabilized formulation containing 10 ppm polysorbate 80.
  • the UV spectrum of the rfurin sample after three hours of agitation shows a slightly elevated profile compared to the sample before agitation, which indicates the presence of aggregates in the agitated sample.
  • the ‘agitated’ spectra is closer to the ‘before agitated’ spectra, relative to those seen in samples without polysorbate ( FIGS. 36 and 37 ), which indicated that that the level of aggregation is less than that seen in samples without polysorbate.
  • FIG. 40 UV spectra of the rfurin samples in the highly stabilized formulation containing 25 ppm polysorbate 80.
  • the UV spectrum of the rfurin sample after three hours of agitation shows a slightly elevated profile compared to the sample before agitation, which indicates presence of a small amount of aggregates in the agitated sample.
  • FIG. 41 UV spectra of the rfurin samples in the highly stabilized formulation containing 50 ppm polysorbate 80.
  • the UV spectrum of the rfurin sample after three hours of agitation shows a very similar profile to the sample before agitation. Neither spectra is slanted or elevated, which indicates that neither sample contains significant amounts of large aggregates.
  • FIG. 42 UV spectra of the rfurin samples in the highly stabilized formulation containing 100 ppm polysorbate 80.
  • the UV spectrum of the rfurin sample after three hours of agitation shows a very similar profile to the sample before agitation. Neither spectra is slanted or elevated, which indicates that neither sample contains significant amounts of large aggregates.
  • FIG. 44 rFurin in the highly stabilized formulation—a freeze/thaw study analyzed using SEC. The same samples as in FIG. 43 . The data show the rfurin peak heights before and after 5 freeze/thaw cycles. The data are sorted from the highest to the lowest.
  • FIG. 45 rFurin in the highly stabilized Formulation—a freeze/thaw study analyzed using SEC. The same samples as in FIG. 44 . The relative peak heights of rfurin are plotted against the number of freeze/thaw cycles. The relative peak heights were calculated as the percentages of the rfurin peak heights before the first freeze/thaw cycle.
  • FIG. 49 Effect of temperature on the pH (B) and conductivity (A) of the highly stabilized formulation stock buffer (500 mM HEPES, 400 mM acetic acid, 1 mM CaCl 2 , pH 6.0).
  • the present disclosure is based in part on the discovery that aqueous furin compositions can be stabilized against various chemical and mechanical stresses by the addition of sugars, sugar alcohols, and non-ionic surfactants.
  • the aqueous furin compositions described herein are significantly more stable when stored at or above room temperature, as well as when subjected to mechanical stress, as compared to similar furin compositions lacking sugars, sugar alcohols, and non-ionic surfactants.
  • the present disclosure is also based in part on the discovery that the addition of non-ionic surfactants to furin dilution buffers enables a greater recovery of furin activity when the enzyme is diluted to lower concentrations used during the manufacture of various recombinant biologics, such as VWF.
  • the studies described herein demonstrate that the addition of sugar or sugar alcohols to a furin composition enhances the stability of the composition when stored over a period of time.
  • the inclusion of as little as 2% sugar or sugar alcohol in an aqueous furin formulation can increase the stability of the composition by greater than 10%.
  • inclusion of 10% sugar or sugar alcohol in an aqueous furin formulation can increase the stability of the composition by greater than 75%.
  • the studies described herein demonstrate that the addition of non-ionic surfactant to a furin composition enhances the stability of the composition when subjected to mechanical stress.
  • the inclusion of as little as 10 ppm of non-ionic surfactant in an aqueous furin formulation can increase the stability of the composition by greater than 75%.
  • the studies described herein demonstrate that the inclusion of non-ionic surfactant to a furin dilution buffer increases the recovery of furin activity after dilution.
  • the inclusion of 75 ppm of non-ionic surfactant in a furin dilution buffer increases the recovery of furin activity after dilution by 3- to 4-fold.
  • compositions of highly stabilized furin formulations are provided.
  • furin recombinant furin
  • any furin protein including furin isolated from a subject or any derivatives or mutants of furin, can be included in the formulations of the present invention.
  • the present disclosure provides highly stabilized furin formulations that show improved stability over control formulations when assayed after subjecting the formulation to one or more stressors, including without limitation exposure of the formulations to a range of temperatures, to several freeze/thaw cycles, and/or to agitation.
  • the present disclosure provides methods for forming highly stabilized furin formulations of the present invention.
  • Such methods include methods for stabilizing a formulation of furin for storage, including frozen storage, storage at ambient room temperature or above, or lyophilization.
  • the present disclosure provides methods for diluting concentrated furin solutions that result in increased retention of furin activity after dilution.
  • aqueous compositions of furin diluted according to the methods provided herein retain 3- to 4-times more enzymatic activity.
  • furin refers to any protein or polypeptide with furin activity, particularly the ability to cleave the peptide bond between residues Arg-763 and Ser-764 of the pro-von Willebrand Factor (pro-VWF) polypeptide.
  • furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to that of NP — 002560.1 (human furin preproprotein).
  • furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to amino acids 25-794 of NP — 002560.1 (human furin proprotein).
  • furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to amino acids 108-794 of NP — 002560.1 (human mature furin protein).
  • furin polypeptides also include natural variants of furin with VWF cleaving activity, as well as modified furin constructs with VWF cleaving activity.
  • furin encompasses any natural variants, alternative sequences, isoforms or mutant proteins that retain some basal activity (for example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more activity as compared to the activity of wild-type furin).
  • furin mutations found in the human population include, without limitation, A43E, A43G, A43V, R50W, Y64C, L77P, R81C, E97A, E97V, V109M, V109L, R130W, A139V, P169T, N245S, E271K, Q339R, N407S, E457Q, R464W, K469R, S524Y, T536S, L570F, D624N, A642T, S685P, V728I, V735I, R745Q, P772L, and A793T.
  • Furin polypeptides also include polypeptides containing post-translational modification. For example, it has been shown that furin is phosphorylated at residues S773 and S775 and predicted that furin is glycosylated at residues N387, N440, and N553.
  • furin proteins include recombinant furin polypeptides as well as native furin polypeptides isolated from a source material (e.g., tissue or blood).
  • Furin polypeptides may be derived from any suitable organism, for example, a mammal such as a primate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster, gerbil, canine, feline, and biologically active derivatives thereof.
  • Mutant and variant furin polypeptides having VWF cleaving activity are also embraced, as are functional fragments, and fusion proteins comprising furin polypeptides.
  • the furin polypeptides described herein may further comprise tags that facilitate purification, detection, or both.
  • the furin polypeptides described herein may further be modified with a therapeutic moiety or a moiety suitable for imaging in vitro or in vivo.
  • Proteolytically active recombinant furin may be prepared by expression in cell culture (e.g., mammalian cell culture).
  • cell culture e.g., mammalian cell culture
  • Non-limiting examples of expression and purification methods for preparing recombinant furin are described in WO 1991/06314, WO 1992/09698, U.S. Pat. Nos. 6,210,929 and 6,596,526, as well as in U.S. Patent Application Publication Nos. 2009/0181423 and 2009/0304669, the contents of which are hereby incorporated by reference in their entireties for all purposes.
  • activity refers to a functional activity or activities of furin or portion thereof associated with a full-length (complete) protein.
  • Substrates for furin include von Willebrand factor, proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, and the beta subunit of pro-nerve growth factor.
  • one Unit (U) of furin activity is defined as the amount of furin that releases 1 pmol of AMC from Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1) per minute.
  • the term “stability” (such as furin stability or furin formulation stability) is used in a structural context, e.g., relating to the structural integrity of a protein, or in a functional context, e.g., relating to a protein's ability to retain its function and/or activity over time.
  • the protein under discussion may be contained within a formulation in accordance with the methods and compositions described herein, and the stability of that protein refers to its stability in that formulation.
  • the stability of a furin composition is determined by measuring the furin activity of the composition.
  • the stability of furin composition formulated with sugar, sugar alcohol, and/or non-ionic surfactant, as described herein, is compared to a furin composition formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.
  • a “storage stable” aqueous furin composition refers to a furin polypeptide solution (e.g., a rfurin polypeptide solution) that has been formulated to increase the stability of the protein in solution, for example by at least 10%, over a given storage time.
  • a furin polypeptide solution e.g., a rfurin polypeptide solution
  • a furin polypeptide solution can be made “storage stable” by the addition of a sugar, sugar alcohol, or non-ionic surfactant as a stabilizing agent.
  • the stability of the furin polypeptide in any given formulation can be measured, for example, by monitoring the formation of aggregates, loss of bulk enzymatic activity, or formation of degradation products, over a period of time.
  • the absolute stability of a formulation, and the stabilizing effects of the sugar, sugar alcohol, or non-ionic surfactant, will vary dependent upon the particular composition being stabilized.
  • the stability of a furin composition is determined by measuring the furin activity of the composition.
  • the stability of furin composition formulated with sugar, sugar alcohol, and/or non-ionic surfactant, as described herein, is compared to a furin composition formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.
  • shelf-life refers to the period of time a formulation maintains a predetermined level of stability at a predetermined temperature.
  • the predetermined temperature refers to frozen (e.g., ⁇ 80° C., ⁇ 25° C., 0° C.), refrigerated (e.g., 0° to 10° C.), or room temperature (e.g., 18° C. to 32° C.) storage.
  • time of stability refers to the length of time a formulation is considered stable.
  • the time of stability for a formulation may refer to the length of time for which the level of protein aggregation and/or degradation in the formulation remains below a certain threshold (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.), and/or the length of time a formulation maintains biological activity above a certain threshold (e.g., 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, etc.) of the amount of activity present in the formulation at the start of the storage period.
  • a certain threshold e.g., 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, etc.
  • a storage stable aqueous composition of a furin polypeptide e.g., rfurin polypeptide
  • a sugar, sugar alcohol, and/or non-ionic surfactant will have a longer time of stability than a composition of the same furin polypeptide formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.
  • a storage stable aqueous composition of a furin polypeptide will have a time of stability that is, for example, at least 10% greater than the time of stability for the same composition formulated in the absence of the sugar, sugar alcohol, and/or non-ionic surfactant, or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% greater, or at least 2 times greater, or at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10 times, or more times greater than the time of stability for the same composition formulated in the absence of the sugar, sugar alcohol, and/or non-ionic surfactant.
  • storage means that a formulation is not immediately administered to a subject or utilized in a production process once prepared, but is kept for a period of time under particular conditions (e.g. particular temperature) prior to use.
  • a furin formulation can be kept for days, weeks, months or years, prior to administration to a subject under varied temperatures such as frozen (e.g., ⁇ 80° C., ⁇ 25° C., 0° C.), refrigerated (e.g., 0° to 10° C.), or room temperature (e.g., 18° C. to 32° C.).
  • such formulations may be liquid or lyophilized formulations.
  • the term “about” denotes an approximate range of plus or minus 10% from a specified value. For instance, the language “about 20%” encompasses a range of 18-22%.
  • the phrases “no more than X” and “from 0 to X” are equivalent and refer to any concentration between and including 0 and X.
  • the phrases “a concentration of no more than 2%” and “a concentration of from 0% to 2%” are equivalent and include 0%, 1%, and 2%.
  • the phrases “no less than X” refers to any concentration X or higher.
  • the phrase “a concentration of no less than 98%” includes 98%, 99%, and 100%.
  • the phrases “between X and Y” and “from X to X” are equivalent and refer to any concentration between and including X and Y.
  • the phrases “a concentration of between 49% and 51%” and “a concentration of from 49% to 51%” are equivalent and include 49%, 50%, and 51%.
  • a “sugar” refers to monosaccharides having the general formula C x H 2y O y (linear) or C x H (2y-1) O y (cyclic), and disaccharides consisting of two monosaccharide units formed through a dehydration reaction. Monosaccharides can be classified by the number of carbon atoms they contain: diose (2), triose (3), tetrose (4), pentose (5), hexose (6), heptose (7), etc. Accordingly, as used herein, a C(X) sugar refers to a sugar containing X-number of carbon molecules.
  • a C(5) sugar refers to a pentose sugar
  • a C(6) sugar refers to a hexose sugar
  • sugars that may be used in the formulations provided herein include: diose sugar glycolaldehyde, triose sugars glyceraldehyde and dihydroxyacetone; tetrose sugars erythrose, threose, and erythrulose; pentose sugars arabinose, lyxose, ribose, xylose, ribulose, and xylulose; hexose sugars allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose; heptose sugars sedoheptulose, mannoheptulose, and L-glycero-D
  • a “sugar alcohol” refers to a hydrogenated form of a mono- or disaccharide, whose carbonyl group has been reduced to form a primary or secondary hydroxyl.
  • the sugar alcohol has between about 4 and about 8 carbon atoms.
  • Non-limiting examples of sugar alcohols that may be used in formulations provided herein include glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • a “pharmaceutically acceptable salt” refers to a salt that is safe for administration to a subject (e.g., a human) in a drug formulation.
  • a subject e.g., a human
  • pharmaceutically acceptable salts are well known in the art, for example, see Stahl and Wermuth, Pharmaceutical Salts Properties, Selection, and Use, 2 nd Revised edition , Wiley, Hoboken, N.J.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
  • non-ionic surfactant refers to a surface active agent that is non-ionized under physiologically relevant conditions.
  • non-limiting examples of non-ionic surfactants useful for the stabilized aqueous furin compositions provided herein include: non-ionic water soluble mono-, di-, and tri-glycerides (e.g., propylene glycol dicarpylate/dicaprate (e.g. MIGLYOL® 840), medium chain mono- and diglycerides (e.g. CAPMUL® and IMWITOR® 72), medium-chain triglycerides (e.g.
  • caprylic and capric triglycerides such as LAVRAFAC, MIGLYOL® 810 or 812, CRODAMOL® GTCC-PN, and SOFTISON 378), long chain monoglycerides (e.g. glyceryl monooleates such as PECEOL®, and glyceryl monolinoleates such as MAISINE®), polyoxyl castor oil (e.g.
  • non-ionic water soluble mono- and di-fatty acid esters of polyethyelene glycol non-ionic water soluble sorbitan fatty acid esters
  • non-ionic water soluble sorbitan fatty acid esters e.g., sorbitan monolaurates such as polyoxyethylene (20) sorbitan monolaurate (TWEEN 20) and sorbitan monolaurate (SPAN 20); sorbitan monopalmitates such as polyoxyethylene (20) sorbitan monopalmitates (TWEEN 40) and sorbitan monopalmitate (SPAN 40); sorbitan monostearates such as polyoxyethylene (20) sorbitan monostearate (TWEEN 60) and sorbitan monostearate (SPAN 60); sorbitan monooleates such as polyoxyethylene (20) sorbitan monooleate (TWEEN 80) and sorbitan monooleate (
  • non-ionic water soluble triblock copolymers e.g. poly(ethyleneoxide)/poly-(propyleneoxide)/poly(ethyleneoxide) triblock copolymers such as methyl-oxirane polymer with oxirane BHT (PLURONIC® F-127)).
  • compositions of furin which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof.
  • a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol,
  • biologically active derivative when used in the context of furin polypeptide, also embraces polypeptides obtained via recombinant DNA technology. This may include any method known in the art for (i) the production of recombinant DNA by genetic engineering, e.g., via reverse transcription of RNA and/or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, e.g., via electroporation or microinjection, (iii) cultivating said transformed cells, e.g., in a continuous or batch-wise manner, (iv) expressing furin protein, e.g., constitutively or upon induction, and (v) isolating said furin protein, e.g., from the culture medium or by harvesting the transformed cells, in order to (vi) obtain substantially purified recombinant furin protein, e.g., via ion exchange chromatography, size exclusion chromat
  • biologically active derivative includes also chimeric molecules such as e.g., a furin protein, or functional fragment thereof, in combination with a second polypeptide, e.g., an immunoglobulin Fc domain or an albumin domain, in order to improve the biological/pharmacological properties such as e.g., half life of the furin protein in the circulation system of a mammal, particularly a human.
  • a second polypeptide e.g., an immunoglobulin Fc domain or an albumin domain
  • BDS refers to “Bulk Drug Substance.”
  • the present disclosure provides stabilized aqueous formulations of furin, e.g., rfurin.
  • furin e.g., rfurin.
  • the following embodiments are based in part on the discovery that inclusion of a sugar, sugar alcohol, and/or non-ionic surfactant stabilizes aqueous furin compositions, as compared to compositions lacking the sugar, sugar alcohol, and/or non-ionic surfactant.
  • furin compositions e.g., rfurin compositions
  • formulated according to the embodiments provided herein may contain, in addition to the components explicitly disclosed, counter ions contributed by the inclusion of solution components or pH modifying agents, for example, sodium or potassium contributed from an acetate salt, sodium hydroxide, or potassium hydroxide or chloride contributed by calcium chloride or hydrochloric acid.
  • a storage stable furin composition e.g., rfurin
  • consisting of or consisting essentially of a given formulation may further comprise one or more counter ion, as necessitated by the formulation process at a particular pH.
  • a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at room temperature (i.e., between 18° C. and 32° C.) for a period of time.
  • room temperature i.e., between 18° C. and 32° C.
  • a storage stable, aqueous immunoglobulin composition will be stable when stored at room temperature for at least 4 days.
  • the composition will be stabile at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days.
  • the composition will be stable for at least 1 month.
  • the composition will be stable for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months.
  • room temperature refers to between 20° C. and 30° C., between 21° C. and 29° C., between 22° C. and 28° C., between 23° C. and 27° C., between 24° C. and 26° C., or about 25° C.
  • the composition will be stable for an extended period of time when stored at a temperature between 20° C. and 25° C.
  • a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at refrigerated temperature (i.e., between 2° C. and 10° C.) for a period of time.
  • refrigerated temperature i.e., between 2° C. and 10° C.
  • a storage stable, aqueous immunoglobulin composition will be stable when stored at refrigerated temperature for at least 4 days.
  • the composition will be stabile at refrigerated temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days.
  • the composition will be stable for at least 1 month.
  • the composition will be stable for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months.
  • the composition will be stable for an extended period of time when stored at a temperature between 2° C. and 8° C.
  • a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at elevated temperature (i.e., between 32° C. and 42° C.) for a period of time.
  • elevated temperature i.e., between 32° C. and 42° C.
  • a storage stable, aqueous immunoglobulin composition will be stable when stored at elevated temperature for at least 4 days.
  • the composition will be stabile at elevated temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days.
  • the composition will be stable for at least 1 month.
  • the composition will be stable for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months.
  • the composition will be stable for an extended period of time when stored at a temperature between 35° C. and 40° C.
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% more furin activity after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • a stabilizing agent e.g., a sugar, sugar alcohol, or non-ionic surfactant
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage more furin activity after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • a stabilizing agent e.g., a sugar, sugar alcohol, or non-ionic surfactant
  • a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 50%. In other embodiments, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% less furin present in an aggregated state after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • a stabilizing agent e.g., a sugar, sugar alcohol, or non-ionic surfactant
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage less furin present in an aggregated state after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • a stabilizing agent e.g., a sugar, sugar alcohol, or non-ionic surfactant
  • the mechanical stress is agitation (e.g., shaking).
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% more furin activity after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • a stabilizing agent e.g., a sugar, sugar alcohol, or non-ionic surfactant
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage more furin activity after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • the mechanical stress is agitation (e.g., shaking).
  • a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 50% after being subjected to mechanical stress. In other embodiments, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after being subjected to mechanical stress.
  • the mechanical stress is agitation (e.g., shaking).
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% less furin present in an aggregated state after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • a stabilizing agent e.g., a sugar, sugar alcohol, or non-ionic surfactant
  • a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage less furin present in an aggregated state after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
  • the mechanical stress is agitation (e.g., shaking).
  • furin (e.g., rfurin) formulations described in this application can be lyophilized and reconstituted in the indicated concentrations, it will be appreciated that these preparations can also be reconstituted in more dilute form.
  • a preparation according the present disclosure which is lyophilized and/or normally reconstituted in 2 mL of solution can also be reconstituted in a larger volume of diluent, such as 5 mL.
  • lyophilized furin (e.g., rfurin) formulations can also be reconstituted in more concentrated form.
  • a preparation according the present disclosure which is lyophilized and/or normally reconstituted in 2 ml of solution can also be reconstituted in a smaller volume, such as 1 mL.
  • the highly stabilized furin (e.g., rfurin) formulations of the present invention are combined with a diluent that confers increased furin recovery when the resultant composition is used in a production method, for example, in the maturation of rVWF (also referred to herein as an “rVWF maturation method”).
  • Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (-Lys-Arg-) at residue 763. It has been shown that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme furin.
  • Production processes for vWF thus include the use of furin, preferably in a highly stabilized formulation.
  • the highly stabilized formulation in this diluent increases furin activity recovery in the rVWF maturation step by three to four times compared to control formulations placed in control diluents.
  • the highly stabilized formulations of the invention have a shelf life of at least 6 months. As will be appreciated, this shelf life may be at frozen temperatures (i.e., ⁇ 80° C., ⁇ 25° C., 0° C.), refrigerated (0° C. to 10° C.), or room temperature (20° C. to 32° C.) in liquid or lyophilized form. In further aspects, the highly stabilized formulations of the invention have a shelf life of at least 12, 18, 24, 30, 36, 42, 48, 54, or 60 months.
  • shelf life is determined by a percent activity remaining after storage at any of the above temperatures for any of the above periods of time.
  • shelf life means that the formulation retains at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% of furin activity as measured by any of the assays described herein or known in the art as compared to activity prior to storage for any of the above amounts of time at any of the above temperatures.
  • a highly stabilized formulation of furin in accordance with the present disclosure includes: (a) 8,000 U/mL-57,000 U/mL furin (e.g., rfurin); (b) 190 mM sodium chloride; (c) 0.92 mM calcium chloride; (d) 10% w/w trehalose dihydrate; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; and (g) 46 mM HEPES.
  • highly stabilized formulations of the present disclosure include the following components: 47 mM HEPES, 46 mM acetic acid, 195 mM sodium chloride, 0.094 mM calcium chloride, 0.0075% polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.
  • highly stabilized formulations of furin, including rfurin include (a) about 5,500 U/mL to 55,000 U/mL; 6,000 U/mL to 50,000 U/mL; 6,500 U/mL to 45,000 U/mL; 7,000 U/mL to 40,000 U/mL; 7,500 U/mL to 35,000 U/mL; 8,000 U/mL to 30,000 U/mL; 8,500 U/mL to 25,000 U/mL; 9,000 U/mL to 20,000 U/mL; 9,500 U/mL to 15,000 U/mL; or 10,000 U/mL furin; (b) about 100 mM to 300 mM, 110 mM to 280 mM, 120 mM to 260 mM, 130 mM to 240 mM, 140 mM to 220 mM, 150 mM to 200 mM, or 160 mM to 180 mM sodium chloride; (c) about 0.5
  • the present disclosure provides a stabilized aqueous furin composition (e.g., rfurin) comprising: furin (e.g., rfurin), from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 10 mM calcium, a buffering agent, and a pH from 5.5 to 7.5.
  • the sugar is a pentose or hexose sugar.
  • a stabilized aqueous furin composition (e.g., rfurin) comprises: furin (e.g., rfurin), from 100 mM to 300 mM of a pharmaceutically acceptable salt; from 0.5 mM to 2 mM calcium; from 2% to 20% of a sugar or sugar alcohol; from 100 to 200 ppm of a non-ionic surfactant; from 10 to 200 mM buffering agent; and a pH from 5.5 to 7.5.
  • the sugar is a pentose or hexose sugar.
  • highly stabilized formulations of the invention include the following components: 47 mM HEPES, 46 mM acetic acid, 195 mM sodium chloride, 0.094 mM calcium chloride, 0.0075% polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.
  • a highly stabilized formulation of furin in accordance with the present invention includes: (a) 8,000-57,000 U/mL rfurin; (b) 190 mM sodium chloride; (c) 0.92 mM calcium chloride; (d) 10% w/w trehalose dihydrate; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; and (g) 46 mM HEPES.
  • aqueous furin e.g., rfurin
  • sugars, sugar alcohols, and non-ionic surfactants stabilizes aqueous furin (e.g., rfurin) compositions.
  • aqueous furin e.g., rfurin
  • these agents increases furin activity retention upon liquid storage, reduces aggregation of furin polypeptides upon liquid storage, reduces the degradation of furin polypeptides upon liquid storage, reduces the loss of furin activity upon agitation of an aqueous furin composition, and reduces aggregation caused by agitation of an aqueous furin composition.
  • the present disclosure provides an aqueous furin composition (e.g., a rfurin composition) comprising from 2% to 20% sugar or sugar alcohol and from 10 ppm to 200 ppm non-ionic surfactant.
  • the composition comprises from 2% to 10% sugar or sugar alcohol and from 10 ppm to 100 ppm non-ionic surfactant.
  • the composition comprises 10 ⁇ 2% sugar or sugar alcohol and 75 ⁇ 25 ppm non-ionic surfactant.
  • the composition comprises 10% sugar or sugar alcohol and 75 ppm non-ionic surfactant.
  • the composition comprises a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9.
  • Var. 434 Var. 519 Var. 604 10-60 Var. 10 Var. 95 Var. 180 Var. 265 Var. 350 Var. 435 Var. 520 Var. 605 10-50 Var. 11 Var. 96 Var. 181 Var. 266 Var. 351 Var. 436 Var. 521 Var. 606 10-25 Var. 12 Var. 97 Var. 182 Var. 267 Var. 352 Var. 437 Var. 522 Var. 607 25-200 Var. 13 Var. 98 Var. 183 Var. 268 Var. 353 Var. 438 Var. 523 Var. 608 25-175 Var. 14 Var. 99 Var. 184 Var. 269 Var. 354 Var. 439 Var. 524 Var. 609 25-150 Var. 15 Var. 100 Var. 185 Var. 270 Var.
  • Var. 440 Var. 525 Var. 610 25-125 Var. 16 Var. 101 Var. 186 Var. 271 Var. 356 Var. 441 Var. 526 Var. 611 25-100 Var. 17 Var. 102 Var. 187 Var. 272 Var. 357 Var. 442 Var. 527 Var. 612 25-90 Var. 18 Var. 103 Var. 188 Var. 273 Var. 358 Var. 443 Var. 528 Var. 613 25-80 Var. 19 Var. 104 Var. 189 Var. 274 Var. 359 Var. 444 Var. 529 Var. 614 25-70 Var. 20 Var. 105 Var. 190 Var. 275 Var. 360 Var. 445 Var. 530 Var. 615 25-60 Var. 21 Var.
  • Var. 632 80 ⁇ 25 Var. 38 Var. 123 Var. 208 Var. 293 Var. 378 Var. 463 Var. 548 Var. 633 90 ⁇ 25 Var. 39 Var. 124 Var. 209 Var. 294 Var. 379 Var. 464 Var. 549 Var. 634 100 ⁇ 25 Var. 40 Var. 125 Var. 210 Var. 295 Var. 380 Var. 465 Var. 550 Var. 635 125 ⁇ 25 Var. 41 Var. 126 Var. 211 Var. 296 Var. 381 Var. 466 Var. 551 Var. 636 150 ⁇ 25 Var. 42 Var. 127 Var. 212 Var. 297 Var. 382 Var. 467 Var. 552 Var. 637 175 ⁇ 25 Var. 43 Var. 128 Var.
  • Var. 643 75 ⁇ 10 Var. 49 Var. 134 Var. 219 Var. 304 Var. 389 Var. 474 Var. 559 Var. 644 80 ⁇ 10 Var. 50 Var. 135 Var. 220 Var. 305 Var. 390 Var. 475 Var. 560 Var. 645 90 ⁇ 10 Var. 51 Var. 136 Var. 221 Var. 306 Var. 391 Var. 476 Var. 561 Var. 646 100 ⁇ 10 Var. 52 Var. 137 Var. 222 Var. 307 Var. 392 Var. 477 Var. 562 Var. 647 110 ⁇ 10 Var. 53 Var. 138 Var. 223 Var. 308 Var. 393 Var. 478 Var. 563 Var. 648 120 ⁇ 10 Var. 54 Var.
  • Var. 665 80 Var. 71 Var. 156 Var. 241 Var. 326 Var. 411 Var. 496 Var. 581 Var. 666 90 Var. 72 Var. 157 Var. 242 Var. 327 Var. 412 Var. 497 Var. 582 Var. 667 100 Var. 73 Var. 158 Var. 243 Var. 328 Var. 413 Var. 498 Var. 583 Var. 668 110 Var. 74 Var. 159 Var. 244 Var. 329 Var. 414 Var. 499 Var. 584 Var. 669 120 Var. 75 Var. 160 Var. 245 Var. 330 Var. 415 Var. 500 Var. 585 Var. 670 125 Var. 76 Var. 161 Var. 246 Var. 331 Var. 416 Var.
  • Var. 422 Var. 507 Var. 592 Var. 677 180 Var. 83 Var. 168 Var. 253 Var. 338 Var. 423 Var. 508 Var. 593 Var. 678 190 Var. 84 Var. 169 Var. 254 Var. 339 Var. 424 Var. 509 Var. 594 Var. 679 200 Var. 85 Var. 170 Var. 255 Var. 340 Var. 425 Var. 510 Var. 595 Var. 680 Var. Variation
  • Var. 1039 Var. 1124 Var. 1209 Var. 1294 25-70 Var. 700 Var. 785 Var.
  • Var. 1215 Var. 1300 50-125 Var. 706 Var. 791 Var. 876 Var. 961 Var. 1046 Var. 1131 Var. 1216 Var. 1301 50-90 Var. 707 Var. 792 Var. 877 Var. 962 Var. 1047 Var. 1132 Var. 1217 Var. 1302 50-80 Var. 708 Var. 793 Var. 878 Var. 963 Var. 1048 Var. 1133 Var. 1218 Var. 1303 75-200 Var. 709 Var. 794 Var. 879 Var. 964 Var. 1049 Var. 1134 Var. 1219 Var. 1304 75-175 Var. 710 Var. 795 Var. 880 Var. 965 Var. 1050 Var. 1135 Var. 1220 Var. 1305 75-150 Var. 711 Var.
  • Var. 1316 150 ⁇ 25 Var. 722 Var. 807 Var. 892 Var. 977 Var. 1062 Var. 1147 Var. 1232 Var. 1317 175 ⁇ 25 Var. 723 Var. 808 Var. 893 Var. 978 Var. 1063 Var. 1148 Var. 1233 Var. 1318 30 ⁇ 10 Var. 724 Var. 809 Var. 894 Var. 979 Var. 1064 Var. 1149 Var. 1234 Var. 1319 40 ⁇ 10 Var. 725 Var. 810 Var. 895 Var. 980 Var. 1065 Var. 1150 Var. 1235 Var. 1320 50 ⁇ 10 Var. 726 Var. 811 Var. 896 Var. 981 Var. 1066 Var. 1151 Var. 1236 Var.
  • Var. 1321 60 ⁇ 10 Var. 727 Var. 812 Var. 897 Var. 982 Var. 1067 Var. 1152 Var. 1237 Var. 1322 70 ⁇ 10 Var. 728 Var. 813 Var. 898 Var. 983 Var. 1068 Var. 1153 Var. 1238 Var. 1323 75 ⁇ 10 Var. 729 Var. 814 Var. 899 Var. 984 Var. 1069 Var. 1154 Var. 1239 Var. 1324 80 ⁇ 10 Var. 730 Var. 815 Var. 900 Var. 985 Var. 1070 Var. 1155 Var. 1240 Var. 1325 90 ⁇ 10 Var. 731 Var. 816 Var. 901 Var. 986 Var. 1071 Var. 1156 Var. 1241 Var. 1326 100 ⁇ 10 Var. 732 Var.
  • Var. 992 Var. 1077 Var. 1162 Var. 1247 Var. 1332 150 ⁇ 10 Var. 738 Var. 823 Var. 908 Var. 993 Var. 1078 Var. 1163 Var. 1248 Var. 1333 160 ⁇ 10 Var. 739 Var. 824 Var. 909 Var. 994 Var. 1079 Var. 1164 Var. 1249 Var. 1334 170 ⁇ 10 Var. 740 Var. 825 Var. 910 Var. 995 Var. 1080 Var. 1165 Var. 1250 Var. 1335 175 ⁇ 10 Var. 741 Var. 826 Var. 911 Var. 996 Var. 1081 Var. 1166 Var. 1251 Var. 1336 180 ⁇ 10 Var. 742 Var. 827 Var. 912 Var. 997 Var. 1082 Var.
  • Var. 1460 Var. 1545 Var. 1630 Var. 1715 Var. 1800 Var. 1885 Var. 1970 25-125 Var. 1376 Var. 1461 Var. 1546 Var. 1631 Var. 1716 Var. 1801 Var. 1886 Var. 1971 25-100 Var. 1377 Var. 1462 Var. 1547 Var. 1632 Var. 1717 Var. 1802 Var. 1887 Var. 1972 25-90 Var. 1378 Var. 1463 Var. 1548 Var. 1633 Var. 1718 Var. 1803 Var. 1888 Var. 1973 25-80 Var. 1379 Var. 1464 Var. 1549 Var. 1634 Var. 1719 Var. 1804 Var. 1889 Var. 1974 25-70 Var. 1380 Var. 1465 Var. 1550 Var. 1635 Var. 1720 Var.
  • Var. 1805 Var. 1890 Var. 1975 25-60 Var. 1381 Var. 1466 Var. 1551 Var. 1636 Var. 1721 Var. 1806 Var. 1891 Var. 1976 25-50 Var. 1382 Var. 1467 Var. 1552 Var. 1637 Var. 1722 Var. 1807 Var. 1892 Var. 1977 50-200 Var. 1383 Var. 1468 Var. 1553 Var. 1638 Var. 1723 Var. 1808 Var. 1893 Var. 1978 50-175 Var. 1384 Var. 1469 Var. 1554 Var. 1639 Var. 1724 Var. 1809 Var. 1894 Var. 1979 50-150 Var. 1385 Var. 1470 Var. 1555 Var. 1640 Var. 1725 Var. 1810 Var. 1895 Var. 1980 50-125 Var. 1386 Var. 1471 Var.
  • Var. 1641 Var. 1726 Var. 1811 Var. 1896 Var. 1981 50-90 Var. 1387 Var. 1472 Var. 1557 Var. 1642 Var. 1727 Var. 1812 Var. 1897 Var. 1982 50-80 Var. 1388 Var. 1473 Var. 1558 Var. 1643 Var. 1728 Var. 1813 Var. 1898 Var. 1983 75-200 Var. 1389 Var. 1474 Var. 1559 Var. 1644 Var. 1729 Var. 1814 Var. 1899 Var. 1984 75-175 Var. 1390 Var. 1475 Var. 1560 Var. 1645 Var. 1730 Var. 1815 Var. 1900 Var. 1985 75-150 Var. 1391 Var. 1476 Var. 1561 Var. 1646 Var. 1731 Var. 1816 Var. 1901 Var.
  • Var. 1510 Var. 1595 Var. 1680 Var. 1765 Var. 1850 Var. 1935 Var. 2020 40 Var. 1426 Var. 1511 Var. 1596 Var. 1681 Var. 1766 Var. 1851 Var. 1936 Var. 2021 50 Var. 1427 Var. 1512 Var. 1597 Var. 1682 Var. 1767 Var. 1852 Var. 1937 Var. 2022 60 Var. 1428 Var. 1513 Var. 1598 Var. 1683 Var. 1768 Var. 1853 Var. 1938 Var. 2023 70 Var. 1429 Var. 1514 Var. 1599 Var. 1684 Var. 1769 Var. 1854 Var. 1939 Var. 2024 75 Var. 1430 Var. 1515 Var. 1600 Var. 1685 Var. 1770 Var. 1855 Var. 1940 Var. 2025 80 Var.
  • Var. 1437 Var. 1522 Var. 1607 Var. 1692 Var. 1777 Var. 1862 Var. 1947 Var. 2032 140 Var. 1438 Var. 1523 Var. 1608 Var. 1693 Var. 1778 Var. 1863 Var. 1948 Var. 2033 150 Var. 1439 Var. 1524 Var. 1609 Var. 1694 Var. 1779 Var. 1864 Var. 1949 Var. 2034 160 Var. 1440 Var. 1525 Var. 1610 Var. 1695 Var. 1780 Var. 1865 Var. 1950 Var. 2035 170 Var. 1441 Var. 1526 Var. 1611 Var. 1696 Var. 1781 Var. 1866 Var. 1951 Var. 2036 175 Var. 1442 Var. 1527 Var. 1612 Var. 1697 Var. 1782 Var. 1867 Var. 1952 Var.
  • Var. 2665 75-150 Var. 2071 Var. 2156 Var. 2241 Var. 2326 Var. 2411 Var. 2496 Var. 2581 Var. 2666 100-200 Var. 2072 Var. 2157 Var. 2242 Var. 2327 Var. 2412 Var. 2497 Var. 2582 Var. 2667 100-175 Var. 2073 Var. 2158 Var. 2243 Var. 2328 Var. 2413 Var. 2498 Var. 2583 Var. 2668 50 ⁇ 25 Var. 2074 Var. 2159 Var. 2244 Var. 2329 Var. 2414 Var. 2499 Var. 2584 Var. 2669 60 ⁇ 25 Var. 2075 Var. 2160 Var. 2245 Var. 2330 Var. 2415 Var. 2500 Var. 2585 Var. 2670 70 ⁇ 25 Var. 2076 Var.
  • Var. 2336 Var. 2421 Var. 2506 Var. 2591 Var. 2676 150 ⁇ 25 Var. 2082 Var. 2167 Var. 2252 Var. 2337 Var. 2422 Var. 2507 Var. 2592 Var. 2677 175 ⁇ 25 Var. 2083 Var. 2168 Var. 2253 Var. 2338 Var. 2423 Var. 2508 Var. 2593 Var. 2678 30 ⁇ 10 Var. 2084 Var. 2169 Var. 2254 Var. 2339 Var. 2424 Var. 2509 Var. 2594 Var. 2679 40 ⁇ 10 Var. 2085 Var. 2170 Var. 2255 Var. 2340 Var. 2425 Var. 2510 Var. 2595 Var. 2680 50 ⁇ 10 Var. 2086 Var. 2171 Var. 2256 Var. 2341 Var. 2426 Var.
  • Var. 2686 100 ⁇ 10 Var. 2092 Var. 2177 Var. 2262 Var. 2347 Var. 2432 Var. 2517 Var. 2602 Var. 2687 110 ⁇ 10 Var. 2093 Var. 2178 Var. 2263 Var. 2348 Var. 2433 Var. 2518 Var. 2603 Var. 2688 120 ⁇ 10 Var. 2094 Var. 2179 Var. 2264 Var. 2349 Var. 2434 Var. 2519 Var. 2604 Var. 2689 125 ⁇ 10 Var. 2095 Var. 2180 Var. 2265 Var. 2350 Var. 2435 Var. 2520 Var. 2605 Var. 2690 130 ⁇ 10 Var. 2096 Var. 2181 Var. 2266 Var. 2351 Var. 2436 Var. 2521 Var. 2606 Var. 2691 140 ⁇ 10 Var.
  • Var. 2459 Var. 2544 Var. 2629 Var. 2714 160 Var. 2120 Var. 2205 Var. 2290 Var. 2375 Var. 2460 Var. 2545 Var. 2630 Var. 2715 170 Var. 2121 Var. 2206 Var. 2291 Var. 2376 Var. 2461 Var. 2546 Var. 2631 Var. 2716 175 Var. 2122 Var. 2207 Var. 2292 Var. 2377 Var. 2462 Var. 2547 Var. 2632 Var. 2717 180 Var. 2123 Var. 2208 Var. 2293 Var. 2378 Var. 2463 Var. 2548 Var. 2633 Var. 2718 190 Var. 2124 Var. 2209 Var. 2294 Var. 2379 Var. 2464 Var. 2549 Var. 2634 Var. 2719 200 Var. 2125 Var. 2210 Var. 2295 Var. 2380 Var. 2465 Var. 2550 Var. 2635 Var. 2720 Var. Variation
  • Var. 3244 Var. 3329 25-150 Var. 2735 Var. 2820 Var. 2905 Var. 2990 Var. 3075 Var. 3160 Var. 3245 Var. 3330 25-125 Var. 2736 Var. 2821 Var. 2906 Var. 2991 Var. 3076 Var. 3161 Var. 3246 Var. 3331 25-100 Var. 2737 Var. 2822 Var. 2907 Var. 2992 Var. 3077 Var. 3162 Var. 3247 Var. 3332 25-90 Var. 2738 Var. 2823 Var. 2908 Var. 2993 Var. 3078 Var. 3163 Var. 3248 Var. 3333 25-80 Var. 2739 Var. 2824 Var. 2909 Var. 2994 Var. 3079 Var. 3164 Var. 3249 Var. 3334 25-70 Var.
  • Var. 2836 Var. 2921 Var. 3006 Var. 3091 Var. 3176 Var. 3261 Var. 3346 100-200 Var. 2752 Var. 2837 Var. 2922 Var. 3007 Var. 3092 Var. 3177 Var. 3262 Var. 3347 100-175 Var. 2753 Var. 2838 Var. 2923 Var. 3008 Var. 3093 Var. 3178 Var. 3263 Var. 3348 50 ⁇ 25 Var. 2754 Var. 2839 Var. 2924 Var. 3009 Var. 3094 Var. 3179 Var. 3264 Var. 3349 60 ⁇ 25 Var. 2755 Var. 2840 Var. 2925 Var. 3010 Var. 3095 Var. 3180 Var. 3265 Var. 3350 70 ⁇ 25 Var. 2756 Var.
  • Var. 2926 Var. 3011 Var. 3096 Var. 3181 Var. 3266 Var. 3351 75 ⁇ 25 Var. 2757 Var. 2842 Var. 2927 Var. 3012 Var. 3097 Var. 3182 Var. 3267 Var. 3352 80 ⁇ 25 Var. 2758 Var. 2843 Var. 2928 Var. 3013 Var. 3098 Var. 3183 Var. 3268 Var. 3353 90 ⁇ 25 Var. 2759 Var. 2844 Var. 2929 Var. 3014 Var. 3099 Var. 3184 Var. 3269 Var. 3354 100 ⁇ 25 Var. 2760 Var. 2845 Var. 2930 Var. 3015 Var. 3100 Var. 3185 Var. 3270 Var. 3355 125 ⁇ 25 Var. 2761 Var. 2846 Var. 2931 Var.
  • Var. 2952 Var. 3037 Var. 3122 Var. 3207 Var. 3292 Var. 3377 190 ⁇ 10 Var. 2783 Var. 2868 Var. 2953 Var. 3038 Var. 3123 Var. 3208 Var. 3293 Var. 3378 25 Var. 2784 Var. 2869 Var. 2954 Var. 3039 Var. 3124 Var. 3209 Var. 3294 Var. 3379 30 Var. 2785 Var. 2870 Var. 2955 Var. 3040 Var. 3125 Var. 3210 Var. 3295 Var. 3380 40 Var. 2786 Var. 2871 Var. 2956 Var. 3041 Var. 3126 Var. 3211 Var. 3296 Var. 3381 50 Var. 2787 Var. 2872 Var. 2957 Var. 3042 Var. 3127 Var. 3212 Var. 3297 Var.
  • Var. 3139 Var. 3224 Var. 3309 Var. 3394 160 Var. 2800 Var. 2885 Var. 2970 Var. 3055 Var. 3140 Var. 3225 Var. 3310 Var. 3395 170 Var. 2801 Var. 2886 Var. 2971 Var. 3056 Var. 3141 Var. 3226 Var. 3311 Var. 3396 175 Var. 2802 Var. 2887 Var. 2972 Var. 3057 Var. 3142 Var. 3227 Var. 3312 Var. 3397 180 Var. 2803 Var. 2888 Var. 2973 Var. 3058 Var. 3143 Var. 3228 Var. 3313 Var. 3398 190 Var. 2804 Var. 2889 Var. 2974 Var. 3059 Var. 3144 Var. 3229 Var. 3314 Var. 3399 200 Var. 2805 Var. 2890 Var. 2975 Var. 3060 Var. 3145 Var. 3230 Var. 3315 Var. 3400 Var. Variation
  • Var. 3405 Var. 3490 Var. 3575 Var. 3660 Var. 3745 Var. 3830 Var. 3915 Var. 4000 10-90 Var. 3406 Var. 3491 Var. 3576 Var. 3661 Var. 3746 Var. 3831 Var. 3916 Var. 4001 10-80 Var. 3407 Var. 3492 Var. 3577 Var. 3662 Var. 3747 Var. 3832 Var. 3917 Var. 4002 10-75 Var. 3408 Var. 3493 Var. 3578 Var. 3663 Var. 3748 Var. 3833 Var. 3918 Var. 4003 10-70 Var.
  • Var. 3494 Var. 3579 Var. 3664 Var. 3749 Var. 3834 Var. 3919 Var. 4004 10-60 Var. 3410 Var. 3495 Var. 3580 Var. 3665 Var. 3750 Var. 3835 Var. 3920 Var. 4005 10-50 Var. 3411 Var. 3496 Var. 3581 Var. 3666 Var. 3751 Var. 3836 Var. 3921 Var. 4006 10-25 Var. 3412 Var. 3497 Var. 3582 Var. 3667 Var. 3752 Var. 3837 Var. 3922 Var. 4007 25-200 Var. 3413 Var. 3498 Var. 3583 Var. 3668 Var. 3753 Var. 3838 Var. 3923 Var. 4008 25-175 Var. 3414 Var. 3499 Var. 3584 Var. 3669 Var. 3754 Var.
  • Var. 4020 50-125 Var. 3426 Var. 3511 Var. 3596 Var. 3681 Var. 3766 Var. 3851 Var. 3936 Var. 4021 50-90 Var. 3427 Var. 3512 Var. 3597 Var. 3682 Var. 3767 Var. 3852 Var. 3937 Var. 4022 50-80 Var. 3428 Var. 3513 Var. 3598 Var. 3683 Var. 3768 Var. 3853 Var. 3938 Var. 4023 75-200 Var. 3429 Var. 3514 Var. 3599 Var. 3684 Var. 3769 Var. 3854 Var. 3939 Var. 4024 75-175 Var. 3430 Var. 3515 Var. 3600 Var. 3685 Var. 3770 Var. 3855 Var. 3940 Var.
  • Var. 4036 150 ⁇ 25 Var. 3442 Var. 3527 Var. 3612 Var. 3697 Var. 3782 Var. 3867 Var. 3952 Var. 4037 175 ⁇ 25 Var. 3443 Var. 3528 Var. 3613 Var. 3698 Var. 3783 Var. 3868 Var. 3953 Var. 4038 30 ⁇ 10 Var. 3444 Var. 3529 Var. 3614 Var. 3699 Var. 3784 Var. 3869 Var. 3954 Var. 4039 40 ⁇ 10 Var. 3445 Var. 3530 Var. 3615 Var. 3700 Var. 3785 Var. 3870 Var. 3955 Var. 4040 50 ⁇ 10 Var. 3446 Var. 3531 Var. 3616 Var. 3701 Var. 3786 Var.
  • Var. 3956 Var. 4041 60 ⁇ 10 Var. 3447 Var. 3532 Var. 3617 Var. 3702 Var. 3787 Var. 3872 Var. 3957 Var. 4042 70 ⁇ 10 Var. 3448 Var. 3533 Var. 3618 Var. 3703 Var. 3788 Var. 3873 Var. 3958 Var. 4043 75 ⁇ 10 Var. 3449 Var. 3534 Var. 3619 Var. 3704 Var. 3789 Var. 3874 Var. 3959 Var. 4044 80 ⁇ 10 Var. 3450 Var. 3535 Var. 3620 Var. 3705 Var. 3790 Var. 3875 Var. 3960 Var. 4045 90 ⁇ 10 Var. 3451 Var. 3536 Var. 3621 Var. 3706 Var. 3791 Var. 3876 Var. 3961 Var.
  • Var. 4057 190 ⁇ 10 Var. 3463 Var. 3548 Var. 3633 Var. 3718 Var. 3803 Var. 3888 Var. 3973 Var. 4058 25 Var. 3464 Var. 3549 Var. 3634 Var. 3719 Var. 3804 Var. 3889 Var. 3974 Var. 4059 30 Var. 3465 Var. 3550 Var. 3635 Var. 3720 Var. 3805 Var. 3890 Var. 3975 Var. 4060 40 Var. 3466 Var. 3551 Var. 3636 Var. 3721 Var. 3806 Var. 3891 Var. 3976 Var. 4061 50 Var. 3467 Var. 3552 Var. 3637 Var. 3722 Var. 3807 Var. 3892 Var. 3977 Var.
  • Var. 4062 60 Var. 3468 Var. 3553 Var. 3638 Var. 3723 Var. 3808 Var. 3893 Var. 3978 Var. 4063 70 Var. 3469 Var. 3554 Var. 3639 Var. 3724 Var. 3809 Var. 3894 Var. 3979 Var. 4064 75 Var. 3470 Var. 3555 Var. 3640 Var. 3725 Var. 3810 Var. 3895 Var. 3980 Var. 4065 80 Var. 3471 Var. 3556 Var. 3641 Var. 3726 Var. 3811 Var. 3896 Var. 3981 Var. 4066 90 Var. 3472 Var. 3557 Var. 3642 Var. 3727 Var. 3812 Var. 3897 Var. 3982 Var. 4067 100 Var. 3473 Var. 3558 Var. 3643 Var. 3728 Var. 3813 Var.
  • Var. 3819 Var. 3904 Var. 3989 Var. 4074 160 Var. 3480 Var. 3565 Var. 3650 Var. 3735 Var. 3820 Var. 3905 Var. 3990 Var. 4075 170 Var. 3481 Var. 3566 Var. 3651 Var. 3736 Var. 3821 Var. 3906 Var. 3991 Var. 4076 175 Var. 3482 Var. 3567 Var. 3652 Var. 3737 Var. 3822 Var. 3907 Var. 3992 Var. 4077 180 Var. 3483 Var. 3568 Var. 3653 Var. 3738 Var. 3823 Var. 3908 Var. 3993 Var. 4078 190 Var. 3484 Var. 3569 Var. 3654 Var. 3739 Var. 3824 Var. 3909 Var. 3994 Var. 4079 200 Var. 3485 Var. 3570 Var. 3655 Var. 3740 Var. 3825 Var. 3910 Var. 3995 Var. 4080 Var. Variation
  • Var. 4689 25-150 Var. 4095 Var. 4180 Var. 4265 Var. 4350 Var. 4435 Var. 4520 Var. 4605 Var. 4690 25-125 Var. 4096 Var. 4181 Var. 4266 Var. 4351 Var. 4436 Var. 4521 Var. 4606 Var. 4691 25-100 Var. 4097 Var. 4182 Var. 4267 Var. 4352 Var. 4437 Var. 4522 Var. 4607 Var. 4692 25-90 Var. 4098 Var. 4183 Var. 4268 Var. 4353 Var. 4438 Var. 4523 Var. 4608 Var. 4693 25-80 Var. 4099 Var. 4184 Var. 4269 Var. 4354 Var. 4439 Var. 4524 Var. 4609 Var. 4694 25-70 Var. 4100 Var. 4185 Var.
  • Var. 4700 50-125 Var. 4106 Var. 4191 Var. 4276 Var. 4361 Var. 4446 Var. 4531 Var. 4616 Var. 4701 50-90 Var. 4107 Var. 4192 Var. 4277 Var. 4362 Var. 4447 Var. 4532 Var. 4617 Var. 4702 50-80 Var. 4108 Var. 4193 Var. 4278 Var. 4363 Var. 4448 Var. 4533 Var. 4618 Var. 4703 75-200 Var. 4109 Var. 4194 Var. 4279 Var. 4364 Var. 4449 Var. 4534 Var. 4619 Var. 4704 75-175 Var. 4110 Var. 4195 Var. 4280 Var. 4365 Var. 4450 Var. 4535 Var. 4620 Var. 4705 75-150 Var. 4111 Var.
  • Var. 4716 150 ⁇ 25 Var. 4122 Var. 4207 Var. 4292 Var. 4377 Var. 4462 Var. 4547 Var. 4632 Var. 4717 175 ⁇ 25 Var. 4123 Var. 4208 Var. 4293 Var. 4378 Var. 4463 Var. 4548 Var. 4633 Var. 4718 30 ⁇ 10 Var. 4124 Var. 4209 Var. 4294 Var. 4379 Var. 4464 Var. 4549 Var. 4634 Var. 4719 40 ⁇ 10 Var. 4125 Var. 4210 Var. 4295 Var. 4380 Var. 4465 Var. 4550 Var. 4635 Var. 4720 50 ⁇ 10 Var. 4126 Var. 4211 Var. 4296 Var. 4381 Var. 4466 Var. 4551 Var. 4636 Var.
  • Var. 4737 190 ⁇ 10 Var. 4143 Var. 4228 Var. 4313 Var. 4398 Var. 4483 Var. 4568 Var. 4653 Var. 4738 25 Var. 4144 Var. 4229 Var. 4314 Var. 4399 Var. 4484 Var. 4569 Var. 4654 Var. 4739 30 Var. 4145 Var. 4230 Var. 4315 Var. 4400 Var. 4485 Var. 4570 Var. 4655 Var. 4740 40 Var. 4146 Var. 4231 Var. 4316 Var. 4401 Var. 4486 Var. 4571 Var. 4656 Var. 4741 50 Var. 4147 Var. 4232 Var. 4317 Var. 4402 Var. 4487 Var. 4572 Var. 4657 Var. 4742 60 Var. 4148 Var. 4233 Var. 4318 Var.
  • Var. 5375 Var. 4781 Var. 4866 Var. 4951 Var. 5036 Var. 5121 Var. 5206 Var. 5291 Var. 5376 25-50 Var. 4782 Var. 4867 Var. 4952 Var. 5037 Var. 5122 Var. 5207 Var. 5292 Var. 5377 50-200 Var. 4783 Var. 4868 Var. 4953 Var. 5038 Var. 5123 Var. 5208 Var. 5293 Var. 5378 50-175 Var. 4784 Var. 4869 Var. 4954 Var. 5039 Var. 5124 Var. 5209 Var. 5294 Var. 5379 50-150 Var. 4785 Var. 4870 Var. 4955 Var. 5040 Var. 5125 Var. 5210 Var. 5295 Var. 5380 50-125 Var. 4786 Var. 4871 Var.
  • Var. 5041 Var. 5126 Var. 5211 Var. 5296 Var. 5381 50-90 Var. 4787 Var. 4872 Var. 4957 Var. 5042 Var. 5127 Var. 5212 Var. 5297 Var. 5382 50-80 Var. 4788 Var. 4873 Var. 4958 Var. 5043 Var. 5128 Var. 5213 Var. 5298 Var. 5383 75-200 Var. 4789 Var. 4874 Var. 4959 Var. 5044 Var. 5129 Var. 5214 Var. 5299 Var. 5384 75-175 Var. 4790 Var. 4875 Var. 4960 Var. 5045 Var. 5130 Var. 5215 Var. 5300 Var. 5385 75-150 Var. 4791 Var. 4876 Var. 4961 Var. 5046 Var. 5131 Var. 5216 Var.
  • Var. 5386 100-200 Var. 4792 Var. 4877 Var. 4962 Var. 5047 Var. 5132 Var. 5217 Var. 5302 Var. 5387 100-175 Var. 4793 Var. 4878 Var. 4963 Var. 5048 Var. 5133 Var. 5218 Var. 5303 Var. 5388 50 ⁇ 25 Var. 4794 Var. 4879 Var. 4964 Var. 5049 Var. 5134 Var. 5219 Var. 5304 Var. 5389 60 ⁇ 25 Var. 4795 Var. 4880 Var. 4965 Var. 5050 Var. 5135 Var. 5220 Var. 5305 Var. 5390 70 ⁇ 25 Var. 4796 Var. 4881 Var. 4966 Var. 5051 Var. 5136 Var. 5221 Var. 5306 Var. 5391 75 ⁇ 25 Var.
  • Var. 4882 Var. 4967 Var. 5052 Var. 5137 Var. 5222 Var. 5307 Var. 5392 80 ⁇ 25 Var. 4798 Var. 4883 Var. 4968 Var. 5053 Var. 5138 Var. 5223 Var. 5308 Var. 5393 90 ⁇ 25 Var. 4799 Var. 4884 Var. 4969 Var. 5054 Var. 5139 Var. 5224 Var. 5309 Var. 5394 100 ⁇ 25 Var. 4800 Var. 4885 Var. 4970 Var. 5055 Var. 5140 Var. 5225 Var. 5310 Var. 5395 125 ⁇ 25 Var. 4801 Var. 4886 Var. 4971 Var. 5056 Var. 5141 Var. 5226 Var. 5311 Var. 5396 150 ⁇ 25 Var. 4802 Var. 4887 Var.
  • Var. 5078 Var. 5163 Var. 5248 Var. 5333 Var. 5418 25 Var. 4824 Var. 4909 Var. 4994 Var. 5079 Var. 5164 Var. 5249 Var. 5334 Var. 5419 30 Var. 4825 Var. 4910 Var. 4995 Var. 5080 Var. 5165 Var. 5250 Var. 5335 Var. 5420 40 Var. 4826 Var. 4911 Var. 4996 Var. 5081 Var. 5166 Var. 5251 Var. 5336 Var. 5421 50 Var. 4827 Var. 4912 Var. 4997 Var. 5082 Var. 5167 Var. 5252 Var. 5337 Var. 5422 60 Var. 4828 Var. 4913 Var. 4998 Var. 5083 Var. 5168 Var. 5253 Var. 5338 Var. 5423 70 Var.
  • Var. 5265 Var. 5350 Var. 5435 170 Var. 4841 Var. 4926 Var. 5011 Var. 5096 Var. 5181 Var. 5266 Var. 5351 Var. 5436 175 Var. 4842 Var. 4927 Var. 5012 Var. 5097 Var. 5182 Var. 5267 Var. 5352 Var. 5437 180 Var. 4843 Var. 4928 Var. 5013 Var. 5098 Var. 5183 Var. 5268 Var. 5353 Var. 5438 190 Var. 4844 Var. 4929 Var. 5014 Var. 5099 Var. 5184 Var. 5269 Var. 5354 Var. 5439 200 Var. 4845 Var. 4930 Var. 5015 Var. 5100 Var. 5185 Var. 5270 Var. 5355 Var. 5440 Var. Variation
  • Var. 5880 Var. 5965 25-125 Var. 5456 Var. 5541 Var. 5626 Var. 5711 Var. 5796 Var. 5881 Var. 5966 25-100 Var. 5457 Var. 5542 Var. 5627 Var. 5712 Var. 5797 Var. 5882 Var. 5967 25-90 Var. 5458 Var. 5543 Var. 5628 Var. 5713 Var. 5798 Var. 5883 Var. 5968 25-80 Var. 5459 Var. 5544 Var. 5629 Var. 5714 Var. 5799 Var. 5884 Var. 5969 25-70 Var. 5460 Var. 5545 Var. 5630 Var. 5715 Var. 5800 Var. 5885 Var. 5970 25-60 Var.
  • Var. 5522 Var. 5607 Var. 5692 Var. 5777 Var. 5862 Var. 5947 Var. 6032 180 Var. 5523 Var. 5608 Var. 5693 Var. 5778 Var. 5863 Var. 5948 Var. 6033 190 Var. 5524 Var. 5609 Var. 5694 Var. 5779 Var. 5864 Var. 5949 Var. 6034 200 Var. 5525 Var. 5610 Var. 5695 Var. 5780 Var. 5865 Var. 5950 Var. 6035 Var. Variation
  • a storage stable composition of furin contains a stabilizing amount of sugar and/or sugar alcohol.
  • the storage stable composition of furin contains monosaccharide sugar.
  • the monosaccharide sugar is selected from the group consisting of a diose sugar, a triose sugar, a tetrose sugar, a pentose sugar, a hexose sugar, a heptose sugar, and an octose sugar.
  • the sugar is a pentose sugar, a hexose sugar, or a combination thereof.
  • the sugar is a hexose sugar.
  • the storage stable composition of furin contains a disaccharide sugar.
  • the disaccharide sugar is selected from disaccharide sugars formed from pentose and/or hexose monosaccharides.
  • the sugar is selected from disaccharide sugars formed from hexose monosaccharides.
  • the sugar is sucrose, trehalose, or a combination thereof.
  • the sugar is sucrose.
  • the sugar is trehalose.
  • the sugar is formulated as trehalose dihydrate.
  • the storage stable composition of furin contains a sugar alcohol.
  • the sugar alcohol is selected from glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol is mannitol.
  • the stable composition of furin contains a mixture of sugar and sugar alcohol.
  • the mixture contains at least two of a monosaccharide, a disaccharide, and a sugar alcohol.
  • the mixture contains at least two of a pentose sugar, a hexose sugar, a disaccharide formed from pentose and/or hexose monosaccharides, and a sugar alcohol.
  • the mixture contains at least two of sucrose, trehalose, and mannitol.
  • the sugar or sugar alcohol is present at a concentration of from 2% to 20%, 2% to 17.5%, 2% to 15%, 2% to 12.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 5% to 20%, 5% to 17.5%, 5% to 15%, 5% to 12.5%, 5% to 10%, 7.5% to 20%, 7.5% to 17.5%, 7.5% to 15%, 7.5% to 12.5%, 10% to 20%, 10% to 17.5%, 10% to 15%, 4 ⁇ 2%, 5 ⁇ 2%, 6 ⁇ 2%, 7 ⁇ 2%, 8 ⁇ 2%, 9 ⁇ 2%, 10 ⁇ 2%, 11 ⁇ 2%, 12 ⁇ 2%, 13 ⁇ 2%, 14 ⁇ 2%, 15 ⁇ 2%, 16 ⁇ 2%, 17 ⁇ 2%, 18 ⁇ 2%, 3 ⁇ 1%, 4 ⁇ 1%, 5 ⁇ 1%, 6 ⁇ 1%, 7 ⁇ 1%, 8 ⁇ 1%, 9 ⁇ 1%, 10 ⁇ 1%, 11 ⁇ 1%, 12 ⁇ 1%, 13 ⁇ 1%, 14 ⁇ 1%, 15 ⁇ 1%, 16 ⁇ 1%, 17 ⁇ 1%, 18 ⁇ 1%, 19 ⁇ 1%, 2%,
  • a stabilized furin (e.g., rfurin) composition comprises from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% trehalose, from 10 ppm to 100 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% trehalose, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10 ⁇ 5% sugar or sugar alcohol, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 10 to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10 ⁇ 2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10 ⁇ 1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10 ⁇ 5% sugar or sugar alcohol, from 10 to 100 ppm non-ionic surfactant, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10 ⁇ 2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10 ⁇ 1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10 ⁇ 5% trehalose, from 10 to 100 ppm non-ionic surfactant, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 10 ⁇ 2% trehalose.
  • the composition contains 10 ⁇ 1% trehalose.
  • the composition contains 10% trehalose.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10 ⁇ 5% sugar or sugar alcohol, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the storage stable furin composition further comprises from 10 to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 10 ⁇ 2% sugar or sugar alcohol.
  • the composition contains 10 ⁇ 1% sugar or sugar alcohol.
  • the composition contains 10% sugar or sugar alcohol.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10 ⁇ 5% sugar or sugar alcohol, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH from of 6.0 ⁇ 0.2.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 10 ⁇ 2% sugar or sugar alcohol.
  • the composition contains 10 ⁇ 1% sugar or sugar alcohol.
  • the composition contains 10% sugar or sugar alcohol.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10 ⁇ 5% trehalose, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 10 ⁇ 2% trehalose.
  • the composition contains 10 ⁇ 1% trehalose.
  • the composition contains 10% trehalose.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • stabilizing agents used in the formulations of the present disclosure are selected from a group that includes, without limitation: sucrose, trehalose, mannitol, raffinose, and arginine. These agents are present in the formulations of the present invention in an amount of from 0.1% to 20%. In certain embodiments, the stabilizing agent is present in an amount of from 5% to 15%, or about 10%.
  • formulations of the present disclosure include stabilizing agents in an amount of from 0.5% to 19%, 1% to 18%, 1.5% to 17%, 2.0% to 16%, 2.5% to 15%, 3.0% to 14%, 3.5% to 13%, 4.0% to 12%, 4.5% to 11%, 5.0% to 10%, 5.5% to 9%, or 6.0% to 8%.
  • Certain formulations include mannitol, sucrose, and/or trehalose in combination with one or more of any other formulation components disclosed herein.
  • a storage stable composition of furin contains a stabilizing amount of non-ionic surfactant.
  • compositions of furin which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof.
  • a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol,
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the non-ionic surfactant is present at a concentration of from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • a stabilized furin (e.g., rfurin) composition comprises from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic water soluble sorbitan fatty acid ester, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and a non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 10 ppm to 200 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic water soluble sorbitan fatty acid ester, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 75 ⁇ 25% non-ionic surfactant, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75 ⁇ 15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ⁇ 5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75 ⁇ 15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ⁇ 5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic water soluble sorbitan fatty acid ester, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 75 ⁇ 15 ppm non-ionic water soluble sorbitan fatty acid ester.
  • the composition contains 75 ⁇ 5 ppm non-ionic water soluble sorbitan fatty acid ester.
  • the composition contains 75 ppm non-ionic water soluble sorbitan fatty acid ester.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 90 ⁇ 25 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 75 ⁇ 25% non-ionic surfactant, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the storage stable furin composition further comprises from 2% to 10% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75 ⁇ 15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ⁇ 5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, 91 mM buffering agent, and a pH from of 6.0 ⁇ 0.2.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 75 ⁇ 15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ⁇ 5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic water soluble sorbitan fatty acid ester, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the composition contains 75 ⁇ 15 ppm non-ionic water soluble sorbitan fatty acid ester.
  • the composition contains 75 ⁇ 5 ppm non-ionic water soluble sorbitan fatty acid ester.
  • the composition contains 75 ppm non-ionic water soluble sorbitan fatty acid ester.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pH of the composition is 6M.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the furin (e.g., rfurin) compositions of the present invention also preferably include a surfactant, preferably a non-ionic surfactant, and preferably in an amount from 0.1 ppm to 150 ppm, or from 65 ppm to 80 ppm, or at about 75 ppm.
  • a surfactant preferably a non-ionic surfactant
  • the furin (e.g., rfurin) compositions include a surfactant in an amount of from 0.5 ppm to 140 ppm, 1.0 ppm to 130 ppm, 10 ppm to 120 ppm, 20 ppm to 110 ppm, 30 ppm to 100 ppm, 40 ppm to 95 ppm, 50 ppm to 90 ppm, 55 ppm to 85 ppm, 60 ppm to 80 ppm, or 70 ppm to 75 ppm.
  • a surfactant in an amount of from 0.5 ppm to 140 ppm, 1.0 ppm to 130 ppm, 10 ppm to 120 ppm, 20 ppm to 110 ppm, 30 ppm to 100 ppm, 40 ppm to 95 ppm, 50 ppm to 90 ppm, 55 ppm to 85 ppm, 60 ppm to 80 ppm, or 70 ppm to 75 ppm.
  • the surfactant is chosen from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, pluronic polyols, glycerol, glucamides (such as Mega 8), tritons, and Brij 35 (polyoxyethylene 23 lauryl ether).
  • pluronic polyols sold under the trade name Pluronic, manufactured by the BASF Wyandotte Corporation
  • Pluronic manufactured by the BASF Wyandotte Corporation
  • Pluronic F-38 of a molecular weight of 5,000 and Pluronic F-68, molecular weight 9,000, both contain (by weight) 80 percent hydrophilic polyoxyethylene groups and 20 percent hydrophobic polyoxypropylene groups.
  • the surfactant is polysorbate 80.
  • the polysorbate 80 is vegetable-derived polysorbate 80.
  • compositions of furin e.g., rfurin
  • a pharmaceutically acceptable salt at a concentration tolerated by the furin polypeptide during storage.
  • the pharmaceutically acceptable salt is a chloride salt.
  • the pharmaceutically acceptable salt is a monovalent chloride salt.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
  • the concentration of pharmaceutically acceptable salt in a stabilized furin (e.g., rfurin) composition provided herein is from 10 mM to 500 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 100 mM to 300 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 150 mM to 250 mM. In yet other embodiments, the concentration of pharmaceutically acceptable salt in the stabilized furin (e.g., rfurin) compositions provided herein is selected from variations 6036 to 6180 found in Table 10.
  • Exemplary pharmaceutically acceptable salt concentrations useful for the stabilization of furin (e.g., rfurin) compositions 10 mM to 500 mM Var. 6036 100 mM to 300 mM Var. 6037 150 mM to 250 mM Var. 6038 50 mM to 500 mM Var. 6039 50 mM to 450 mM Var. 6040 50 mM to 400 mM Var. 6041 100 to 400 mM Var. 6042 100 mM to 350 mM Var. 6043 100 mM to 250 mM Var. 6044 100 mM to 200 mM Var. 6045 150 mM to 400 mM Var. 6046 150 mM to 350 mM Var.
  • furin e.g., rfurin
  • Var. 6047 150 mM to 300 mM Var. 6048 150 mM to 250 mM Var. 6049 150 mM to 200 mM Var. 6050 175 mM to 225 mM Var. 6051 175 mM to 200 mM Var. 6052 100 ⁇ 25 mM Var. 6053 150 ⁇ 25 mM Var. 6054 200 ⁇ 25 mM Var. 6055 250 ⁇ 25 mM Var. 6056 300 ⁇ 25 mM Var. 6057 350 ⁇ 25 mM Var. 6058 400 ⁇ 25 mM Var. 6059 450 ⁇ 25 mM Var. 6060 190 ⁇ 150 mM Var. 6061 190 ⁇ 125 mM Var.
  • a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
  • pharmaceutically acceptable salt is sodium chloride.
  • pharmaceutically acceptable salt is potassium chloride.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
  • pharmaceutically acceptable salt is sodium chloride.
  • pharmaceutically acceptable salt is potassium chloride.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 2 mM calcium, 10 ⁇ 5% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
  • pharmaceutically acceptable salt is sodium chloride.
  • pharmaceutically acceptable salt is potassium chloride.
  • the concentration of the buffering agent is 90 ⁇ 25 mM.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, from 91 mM buffering agent, and a pH of 6.0 ⁇ 0.2.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
  • pharmaceutically acceptable salt is sodium chloride.
  • pharmaceutically acceptable salt is potassium chloride.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • monovalent chloride salts such as sodium chloride and potassium chloride
  • formulations of the present disclosure are used in formulations of the present disclosure.
  • the formulation component comprising monovalent chloride salts is described primarily in terms of sodium chloride, it will be appreciated that any monovalent chloride salt, including potassium chloride, may be used in accordance with the descriptions herein for sodium chloride.
  • sodium chloride is included in the present formulations at an amount of from 50 to 500 mM. In other embodiments, sodium chloride is included in the formulations at an amount of from 100 to 300 mM, from 150 to 250 mM, or at about 190 mM.
  • sodium chloride is included in the present formulations in an amount of from 100 to 300 mM, 110 to 280 mM, 120 to 260 mM, 130 to 240 mM, 140 to 220 mM, 150 to 200 mM, or 160 to 180 mM.
  • the formulation includes 190 mM sodium chloride in combination with one or more of any other formulation components disclosed herein.
  • furin (e.g., rfurin) compositions are stabilized at pH 6.0 to 7.0.
  • furin compositions formulated at pH 5.5 are stabilized as compared to furin compositions formulated at pH 5.0.
  • furin compositions formulated at pH 6.0 are further stabilized as compared to compositions formulated at pH 5.5.
  • furin (e.g., rfurin) compositions formulated at pH 7.0 have similar stability to compositions formulated at pH 6.0.
  • the stabilized furin (e.g., rfurin) compositions provided herein are formulated at a pH from 5.5 to 7.5.
  • a stabilized furin (e.g., rfurin) composition is formulated at a pH from 6.0 to 7.0.
  • a stabilized furin composition is formulated at pH 6.0 ⁇ 0.2.
  • a stabilized furin composition is formulated at pH 6.0.
  • a stabilized furin composition is formulated at pH selected from variations 6181 to 6403 found in Table 11.
  • Var. 6199 5.5-6.0 Var. 6200 5.6-8.0 Var. 6201 5.6-7.9 Var. 6202 5.6-7.8 Var. 6203 5.6-7.6 Var. 6204 5.6-7.5 Var. 6205 5.6-7.4 Var. 6206 5.6-7.3 Var. 6207 5.6-7.2 Var. 6208 5.6-7.1 Var. 6209 5.6-7.0 Var. 6210 5.6-6.9 Var. 6211 5.6-6.8 Var. 6212 5.6-6.7 Var. 6213 5.6-6.6 Var. 6214 5.6-6.5 Var. 6215 5.6-6.4 Var. 6216 5.6-6.3 Var. 6217 5.6-6.2 Var. 6218 5.6-6.1 Var. 6219 5.6-6.0 Var. 6220 5.7-8.0 Var.
  • Var. 6222 5.7-7.8 Var. 6223 5.7-7.6 Var. 6224 5.7-7.5 Var. 6225 5.7-7.4 Var. 6226 5.7-7.3 Var. 6227 5.7-7.2 Var. 6228 5.7-7.1 Var. 6229 5.7-7.0 Var. 6230 5.7-6.9 Var. 6231 5.7-6.8 Var. 6232 5.7-6.7 Var. 6233 5.7-6.6 Var. 6234 5.7-6.5 Var. 6235 5.7-6.4 Var. 6236 5.7-6.3 Var. 6237 5.7-6.2 Var. 6238 5.7-6.1 Var. 6239 5.7-6.0 Var. 6240 5.8-8.0 Var. 6241 5.8-7.9 Var.
  • Var. 6242 5.8-7.8 Var. 6243 5.8-7.6 Var. 6244 5.8-7.5 Var. 6245 5.8-7.4 Var. 6246 5.8-7.3 Var. 6247 5.8-7.2 Var. 6248 5.8-7.1 Var. 6249 5.8-7.0 Var. 6250 5.8-6.9 Var. 6251 5.8-6.8 Var. 6252 5.8-6.7 Var. 6253 5.8-6.6 Var. 6254 5.8-6.5 Var. 6255 5.8-6.4 Var. 6256 5.8-6.3 Var. 6257 5.8-6.2 Var. 6258 5.8-6.1 Var. 6259 5.8-6.0 Var. 6260 5.9-8.0 Var. 6261 5.9-7.9 Var. 6262 5.9-7.8 Var.
  • Var. 6263 5.9-7.6 Var. 6264 5.9-7.5 Var. 6265 5.9-7.4 Var. 6266 5.9-7.3 Var. 6267 5.9-7.2 Var. 6268 5.9-7.1 Var. 6269 5.9-7.0 Var. 6270 5.9-6.9 Var. 6271 5.9-6.8 Var. 6272 5.9-6.7 Var. 6273 5.9-6.6 Var. 6274 5.9-6.5 Var. 6275 5.9-6.4 Var. 6276 5.9-6.3 Var. 6277 5.9-6.2 Var. 6278 5.9-6.1 Var. 6279 5.9-6.0 Var. 6280 6.0-8.0 Var. 6281 6.0-7.9 Var. 6282 6.0-7.8 Var. 6283 6.0-7.6 Var.
  • Var. 6284 6.0-7.5 Var. 6285 6.0-7.4 Var. 6286 6.0-7.3 Var. 6287 6.0-7.2 Var. 6288 6.0-7.1 Var. 6289 6.0-7.0 Var. 6290 6.0-6.9 Var. 6291 6.0-6.8 Var. 6292 6.0-6.7 Var. 6293 6.0-6.6 Var. 6294 6.0-6.5 Var. 6295 6.0-6.4 Var. 6296 6.0-6.3 Var. 6297 6.0-6.2 Var. 6298 6.0-6.1 Var. 6299 5.7 ⁇ 0.2 Var. 6300 5.8 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9 ⁇ 0.2 Var. 6301 5.9
  • Var. 6338 7.3 ⁇ 0.1 Var. 6339 7.4 ⁇ 0.1 Var. 6340 7.5 ⁇ 0.1 Var. 6341 7.6 ⁇ 0.1 Var. 6342 7.7 ⁇ 0.1 Var. 6343 7.8 ⁇ 0.1 Var. 6344 5.5 Var. 6345 5.6 Var. 6346 5.7 Var. 6347 5.8 Var. 6348 5.9 Var. 6349 6 Var. 6350 6.1 Var. 6351 6.2 Var. 6352 6.3 Var. 6353 6.4 Var. 6354 6.5 Var. 6355 6.6 Var. 6356 6.7 Var. 6357 6.8 Var. 6358 6.9 Var. 6359 7 Var. 6360 7.1 Var. 6361 7.2 Var.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10 ⁇ 5% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10 ⁇ 5% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, from 90 ⁇ 25 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 191 mM of a pharmaceutically acceptable salt, 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • the stabilized furin (e.g., rfurin) compositions provided herein are formulated at an appropriate pH using one or more buffering agents.
  • the one or more buffering agent is selected from histidine, imidazole, phosphate, citrate, Tris, acetate (e.g., acetic acid), BIS-Tris Propane, PIPES, MOPS, HEPES, MES, ACES, and a combination thereof.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • Buffering agent is present in the stabilized furin (e.g., rfurin) compositions at a concentration suitable to maintain the pH of the composition during storage over a period of time (e.g., weeks, months, or years).
  • the concentration of buffering agent in the formulation is from 10 mM to 300 mM.
  • the concentration of buffering agent in the formulation is between 10 mM to 200 mM.
  • the concentration of buffering agent in the formulation is 90 ⁇ 25 mM.
  • the concentration of buffering agent in the composition is selected from variations 6404 to 6469 found in Table 12.
  • Exemplary buffering agent concentrations (mM) useful for the stabilization of furin (e.g., rfurin) compositions 10-300 Var. 6404 10-275 Var. 6405 10-250 Var. 6406 10-225 Var. 6407 10-200 Var. 6408 10-175 Var. 6409 10-150 Var. 6410 10-125 Var. 6411 10-100 Var. 6412 25-300 Var. 6413 25-275 Var. 6414 25-250 Var. 6415 25-225 Var. 6416 25-200 Var. 6417 25-175 Var. 6418 25-150 Var. 6419 25-125 Var. 6420 25-100 Var. 6421 50-300 Var. 6422 50-275 Var. 6423 50-250 Var. 6424 50-225 Var. 6425 50-200 Var.
  • Var. 6426 50-175 Var. 6427 50-150 Var. 6428 50-125 Var. 6429 50-100 Var. 6430 75-300 Var. 6431 75-275 Var. 6432 75-250 Var. 6433 75-225 Var. 6434 75-200 Var. 6435 75-175 Var. 6436 75-150 Var. 6437 75-125 Var. 6438 75-100 Var. 6439 90 ⁇ 75 Var. 6440 90 ⁇ 50 Var. 6441 90 ⁇ 25 Var. 6442 90 ⁇ 10 Var. 6443 90 ⁇ 5 Var. 6444 90 ⁇ 2 Var. 6445 91 ⁇ 10 Var. 6446 91 ⁇ 5 Var. 6447 91 ⁇ 2 Var. 6448 80 Var. 6449 81 Var. 6450 82 Var. 6451 83 Var.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant.
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10 ⁇ 5% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 190 ⁇ 50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10 ⁇ 5% sugar or sugar alcohol, 75 ⁇ 25 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • a stabilized furin (e.g., rfurin) composition comprises 191 mM of a pharmaceutically acceptable salt, 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5.
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof.
  • the sugar or sugar alcohol is trehalose.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose.
  • the buffering agent is acetate, HEPES, or a combination thereof.
  • the buffering agent is a combination of acetate and HEPES.
  • the pH of the composition is from 5.5 to 7.0.
  • the pH of the composition is from 5.5 to 6.5.
  • the pH of the composition is 6.0 ⁇ 0.2.
  • the pH of the composition is 6.0.
  • the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
  • Buffers may also be present in formulations of the invention in combination with one or more of any other formulation components described herein.
  • the highly stabilized formulations of the present invention show increased stability at pH 6.0.
  • the pH of the highly stabilized formulations should preferably be maintained in the range of from 6.0 to 8.0, or at a pH of about 6.0.
  • the buffering agent can be any physiologically acceptable chemical entity or combination of chemical entities which have the capacity to act as buffers, including, without limitation: histidine, imidazole, phosphate, citrate, Tris, Acetate, BIS-Tris Propane, PIPES, MOPS, HEPES, MES, and ACES. The full chemical designations of many of these buffering agents are listed in Table 1 below.
  • phosphate is not used as a buffering agent.
  • the buffering agent is included in a concentration of from 10 mM to 200 mM, or 10 to 100 mM, or 30-60 mM, or about 46 mM.
  • an individual buffering agent is included in a concentration of from 15 to 95 mM, 20 to 90 mM, 25 to 85 mM, 30 to 80 mM, 35 to 75 mM, 40 to 70 mM, 45 to 65 mM, or 50 to 60 mM.
  • the formulation contains two buffering agents.
  • the present formulations include a carboxylic acid in combination with one or more of the formulation components described herein.
  • the carboxylic acid is preferably acetic acid (e.g., acetate).
  • the formulation includes from 20 mM to 100 mM acetic acid, or from 30 mM to 50 mM, or about 45 mM acetic acid or any other carboxylic acid.
  • the formulation includes from 25 mM to 90 mM, 30 mM to 80 mM, 35 mM to 70 mM, 40 mM to 60 mM, or 45 mM to 50 mM acetic acid or any other carboxylic acid.
  • the highly stabilized formulations of the present disclosure include furin, e.g., rfurin, and one or more of stabilizing agents, buffering agents, sodium chloride, salts, and other excipients. Such components are described in further detail below. As will be appreciated, any of the formulation components described herein can be used singly or in any combination.
  • the highly stabilized Furin formulations of the present disclosure show increased stability toward shear (agitation), lyophilization, and freeze/thaw stress as well as resistance to product loss or denaturation at container surfaces as compared to control or starting formulations.
  • Furin is included in formulations of the present disclosure in concentrations from 5,000 to 500,000 U/mL. In certain embodiments, Furin is included in concentrations from 5,500 to 55,000, from 6,000 to 50,000, from 6,500 to 45,000, from 7,000 to 40,000, from 7,500 to 35,000, from 8,000 to 30,000, from 8,500 to 25,000, from 9,000 to 20,000, from 9,500 to 15,000, and about 10,000 U/mL. In yet other embodiments, furin is included in the formulations of the present disclosure at a concentration selected from variations 6470 to 6533 found in Table 14, reported in thousands of Units of furin activity per mL. In specific embodiments, the furin contained in the highly stabilized formulations of the present disclosure is rfurin.
  • the furin (e.g., rfurin) concentration of a stabilized composition provided herein can be expressed as the mass of furin present in the composition.
  • furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 100 ng/mL to 100 mg/mL.
  • furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 1 ⁇ g/mL to 10 mg/mL.
  • furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 1 ⁇ g/mL to 1 mg/mL.
  • furin e.g., rfurin
  • a stabilized composition as described herein at a concentration selected from variations 6534 to 6638 found in Table 15.
  • the furin contained in the highly stabilized formulations of the present disclosure is rfurin.
  • furin e.g., rfurin
  • concentrations used in the stabilized compositions provided herein. 100 ng/mL to 100 mg/mL Var. 6534 100 ng/mL to 50 mg/mL Var. 6535 100 ng/mL to 25 mg/mL Var. 6536 100 ng/mL to 10 mg/mL Var. 6537 100 ng/mL to 5 mg/mL Var. 6538 100 ng/mL to 1 mg/mL Var. 6539 100 ng/mL to 500 ⁇ g/mL Var. 6540 100 ng/mL to 250 ⁇ g/mL Var. 6541 100 ng/mL to 100 ⁇ g/mL Var.
  • furin e.g., rfurin
  • Var. 6542 100 ng/mL to 50 ⁇ g/mL Var. 6543 100 ng/mL to 25 ⁇ g/mL Var. 6544 100 ng/mL to 10 ⁇ g/mL Var. 6545 100 ng/mL to 5 ⁇ g/mL Var. 6546 100 ng/mL to 1 ⁇ g/mL Var. 6547 1 ⁇ g/mL to 100 mg/mL Var. 6548 1 ⁇ g/mL to 50 mg/mL Var. 6549 1 ⁇ g/mL to 25 mg/mL Var. 6550 1 ⁇ g/mL to 10 mg/mL Var. 6551 1 ⁇ g/mL to 5 mg/mL Var. 6552 1 ⁇ g/mL to 1 mg/mL Var.
  • Var. 6553 1 ⁇ g/mL to 500 ⁇ g/mL
  • Var. 6554 1 ⁇ g/mL to 250 ⁇ g/mL
  • Var. 6555 1 ⁇ g/mL to 100 ⁇ g/mL
  • Var. 6556 1 ⁇ g/mL to 50 ⁇ g/mL
  • Var. 6557 1 ⁇ g/mL to 25 ⁇ g/mL
  • Var. 6558 1 ⁇ g/mL to 10 ⁇ g/mL
  • Var. 6559 1 ⁇ g/mL to 5 ⁇ g/mL
  • Var. 6560 5 ⁇ g/mL to 100 mg/mL
  • Var. 6561 5 ⁇ g/mL to 50 mg/mL
  • Var. 6562 5 ⁇ g/mL to 25 mg/mL Var.
  • Var. 6563 5 ⁇ g/mL to 10 mg/mL Var. 6564 5 ⁇ g/mL to 5 mg/mL Var. 6565 5 ⁇ g/mL to 1 mg/mL Var. 6566 5 ⁇ g/mL to 500 ⁇ g/mL Var. 6567 5 ⁇ g/mL to 250 ⁇ g/mL Var. 6568 5 ⁇ g/mL to 100 ⁇ g/mL Var. 6569 5 ⁇ g/mL to 50 ⁇ g/mL Var. 6570 5 ⁇ g/mL to 25 ⁇ g/mL Var. 6571 5 ⁇ g/mL to 10 ⁇ g/mL Var. 6572 10 ⁇ g/mL to 100 mg/mL Var.
  • Var. 6573 10 ⁇ g/mL to 50 mg/mL Var. 6574 10 ⁇ g/mL to 25 mg/mL Var. 6575 10 ⁇ g/mL to 10 mg/mL Var. 6576 10 ⁇ g/mL to 5 mg/mL Var. 6577 10 ⁇ g/mL to 1 mg/mL Var. 6578 10 ⁇ g/mL to 500 ⁇ g/mL Var. 6579 10 ⁇ g/mL to 250 ⁇ g/mL Var. 6580 10 ⁇ g/mL to 100 ⁇ g/mL Var. 6581 10 ⁇ g/mL to 50 ⁇ g/mL Var. 6582 10 ⁇ g/mL to 25 ⁇ g/mL Var.
  • Var. 6583 25 ⁇ g/mL to 100 mg/mL Var. 6584 25 ⁇ g/mL to 50 mg/mL Var. 6585 25 ⁇ g/mL to 25 mg/mL Var. 6586 25 ⁇ g/mL to 10 mg/mL Var. 6587 25 ⁇ g/mL to 5 mg/mL Var. 6588 25 ⁇ g/mL to 1 mg/mL Var. 6589 25 ⁇ g/mL to 500 ⁇ g/mL Var. 6590 25 ⁇ g/mL to 250 ⁇ g/mL Var. 6591 25 ⁇ g/mL to 100 ⁇ g/mL Var. 6592 25 ⁇ g/mL to 50 ⁇ g/mL Var.
  • Var. 6593 50 ⁇ g/mL to 100 mg/mL Var. 6594 50 ⁇ g/mL to 50 mg/mL Var. 6595 50 ⁇ g/mL to 25 mg/mL Var. 6596 50 ⁇ g/mL to 10 mg/mL Var. 6597 50 ⁇ g/mL to 5 mg/mL Var. 6598 50 ⁇ g/mL to 1 mg/mL Var. 6599 50 ⁇ g/mL to 500 ⁇ g/mL Var. 6600 50 ⁇ g/mL to 250 ⁇ g/mL Var. 6601 50 ⁇ g/mL to 100 ⁇ g/mL Var. 6602 100 ⁇ g/mL to 100 mg/mL Var.
  • Var. 6603 100 ⁇ g/mL to 50 mg/mL Var. 6604 100 ⁇ g/mL to 25 mg/mL Var. 6605 100 ⁇ g/mL to 10 mg/mL Var. 6606 100 ⁇ g/mL to 5 mg/mL Var. 6607 100 ⁇ g/mL to 1 mg/mL Var. 6608 100 ⁇ g/mL to 500 ⁇ g/mL Var. 6609 100 ⁇ g/mL to 250 ⁇ g/mL Var. 6610 250 ⁇ g/mL to 100 mg/mL Var. 6611 250 ⁇ g/mL to 50 mg/mL Var. 6612 250 ⁇ g/mL to 25 mg/mL Var.
  • furin (e.g., rfurin) formulations include calcium or another divalent metal cations.
  • the divalent cation is present as a salt, preferably a chloride salt. In certain embodiments, from 0.1 mM to 10 mM of a divalent cation salt can be used, or from 0.5 mM to 2 mM, or about 0.92 mM.
  • divalent cation salt is used in formulations of the invention.
  • calcium salt is preferably calcium chloride, but can also be other calcium salts such as calcium gluconate, calcium glubionate, or calcium gluceptate.
  • Divalent cations, including calcium can be included in combination with one or more other formulation components disclosed herein.
  • Non-limiting examples of divalent metal cations useful in the formulations provided herein include calcium, barium, manganese, magnesium, cobalt, copper, nickel, and zinc.
  • the furin (e.g., rfurin) formulations include an antioxidant.
  • the addition of antioxidants to aqueous and lyophilized formulations has been found to improve the stability of these formulations, and thus extend their shelf lives.
  • the antioxidants used must be compatible for use with a pharmaceutical preparation, and in addition are preferably water soluble.
  • When adding antioxidants to a formulation it is preferable to add such antioxidants as late in the process prior to lyophilization as possible, in order to avoid spontaneous oxidation of the antioxidant.
  • the furin (e.g., rfurin) composition comprises from 0.05 mg/mL to 1.0 mg/mL antioxidant.
  • the furin (e.g., rfurin) composition comprises from 0.05 to 0.5 mg/mL, 0.1 mg/mL to 0.9 mg/mL, 0.1 mg/mL to 0.8 mg/mL, 0.1 mg/mL to 0.7 mg/mL, 0.1 mg/mL to 0.6 mg/mL, 0.1 mg/mL to 0.5 mg/mL, 0.1 mg/mL to 0.4 mg/mL, 0.1 mg/mL to 0.3 mg/mL, 0.1 mg/mL to 0.2 mg/mL, 0.2 mg/mL to 0.9 mg/mL, 0.2 mg/mL to 0.8 mg/mL, 0.2 mg/mL to 0.7 mg/mL, 0.2 mg/mL to 0.6 mg/mL, 0.2 mg/mL to 0.5 mg/mL, 0.2 mg/mL to 0.4 mg/mL, 0.2 mg/mL to 0.3 mg/mL, 0.3 mg/mL, 0.1 mg/mL to 0.2 mg
  • highly stabilized formulations of furin are prepared by providing a starting (control) furin formulation and adding components to achieve a desired level of component concentrations. For example, the addition of one part 750 ppm non-ionic surfactant to 9 parts furin composition lacking non-ionic surfactant to provide a final formulation comprising 75 ppm non-ionic surfactant. This process is also called “spiking in” to a starting formulation.
  • furin is contained in a starting formulation and a buffer composition is spiked into the starting formulation.
  • the highly stabilized furin (e.g., rfurin) formulation is formed using dialysis according to methods known in the art.
  • the methods of formulation involve spiking a concentrated buffer composition into a starting formulation of furin.
  • polysorbate 80 is added to the starting formulation prior to the addition of other components, particularly prior to the addition of trehalose, in order to protect Furin against agitation during mixing.
  • furin (e.g., rfurin) bulk drug substance (BDS) is contained in a starting formulation of 10 mM sodium acetate, 230 mM sodium chloride, 1 mM calcium chloride, pH 6.0.
  • the following components are spiked into the starting formulation: 1% polysorbate 80, 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl 2 , pH 6.0 and trehalose dihydrate powder to attain the following composition: 46 mM HEPES, 45 mM acetic acid, 190 mM NaCl, 0.92 mM CaCl2, 75 ppm polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.
  • the polysorbate 80 is added to the starting formulation prior to the other components to protect furin from aggregation and adsorption to container surfaces during mixing when the other reagents are added.
  • the furin in the starting formulation is mixed with polysorbate 80 and/or any other components except for trehalose. Trehalose and any remaining components are added after polysorbate 80 is added to protect furin from aggregation and adsorption during mixing.
  • the stabilized formulation is combined with a diluent so that the formulation can be used in methods such as an rVWF maturation process.
  • a stabilized diluent is prepared by spiking a starting diluent (50 mM HEPES, 150 mM NaCl, 1 mM CaCl 2 , pH 7.0) with polysorbate 80 to 75 ppm.
  • the combination of the highly stabilized furin (e.g., rfurin) formulation of the present disclosure with this stabilized diluent increases furin recovery in the rVWF maturation step by three to four times compared to the use of the starting furin and diluent formulations.
  • the highly stabilized furin formulations of the present disclosure are lyophilized.
  • the formulation is converted from being in an aqueous phase to being in an amorphous solid phase, which is thought to protect the protein from chemical and/or conformational instability.
  • the lyophilized preparation not only contains an amorphous phase, but also includes a component which crystallizes during lyophilization.
  • One or more components of highly stabilized formulations may in some embodiments be dispersed in the amorphous phase of the lyophilized cake.
  • the apparent glass transition temperature (Tg′) of the amorphous phase is preferably relatively high during freeze-drying, and the glass transition temperature (Tg) of the solid is likewise preferably high during storage.
  • the present disclosure provides methods for diluting aqueous compositions of furin (e.g., rfurin).
  • furin e.g., rfurin
  • the following embodiments are based in part on the discovery that inclusion of a non-ionic surfactant in a buffer used to dilute furin (e.g., rfurin) results in the recovery of 3-4 times more furin activity as compared to dilution with a buffer lacking the non-ionic surfactant.
  • the method includes adding a dilution buffer containing non-ionic surfactant to a furin (e.g., rfurin) composition, to form a diluted furin (e.g., rfurin) composition.
  • a dilution buffer containing non-ionic surfactant to a furin (e.g., rfurin) composition
  • the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 1,000:1 (dilution buffer:furin composition).
  • the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 500:1 (dilution buffer:furin composition).
  • the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 250:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 200:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 100:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 50:1 (dilution buffer:furin composition).
  • the method comprises a first step of adding a non-ionic surfactant to a furin (e.g., rfurin) composition and a second step of adding a dilution buffer to the furin (e.g., rfurin) composition containing the non-ionic surfactant, to form a diluted furin composition.
  • the surfactant will be added to the furin composition at a final concentration of X-fold a desired concentration in the diluted furin composition, where X is the dilution factor.
  • the non-ionic surfactant is added to the starting furin composition at a final concentration of 1,000 ppm (10 ppm ⁇ 100-fold dilution), and the composition is subsequently diluted by adding 99 parts dilution buffer per 1 part starting solution (accounting for volume added during addition of the non-ionic surfactant).
  • the furin (e.g., rfurin) composition is diluted from 1-fold to 1,000-fold, from 1-fold to 500-fold, from 1-fold to 250-fold, from 1-fold to 200-fold, from 1-fold to 100-fold, from 1-fold to 50-fold, from 1-fold to 10-fold, from 10-fold to 1,000-fold, from 10-fold to 500-fold, from 10-fold to 250-fold, from 10-fold to 200-fold, from 10-fold to 100-fold, from 10-fold to 50-fold, from 50-fold to 1,000-fold, from 50-fold to 500-fold, from 50-fold to 250-fold, from 50-fold to 200-fold, from 50-fold to 100-fold, from 100-fold to 1,000-fold, from 100-fold to 500-fold, from 100-fold to 250-fold, from 100-fold to 200-fold, from 200-fold to 1,000-fold, from 200-fold to 500-fold, or from 200-fold to 250-fold.
  • the furin dilution buffer will include a pharmaceutically acceptable salt, non-ionic surfactant, and a buffering agent.
  • the dilution buffer further includes calcium.
  • the dilution buffer further includes a sugar and/or sugar alcohol.
  • pharmaceutically acceptable salt is present in the dilution buffer at a concentration of from 10 mM to 500 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 100 mM to 300 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 150 mM to 250 mM. In yet other embodiments, the concentration of pharmaceutically acceptable salt in the furin dilution buffer is selected from variations 6036 to 6180 found in Table 10.
  • the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride.
  • the dilution buffer has a pH of from 5.5 to 7.5. In one embodiment, the pH of the dilution buffer is from 5.5 to 7.0. In another embodiment, the pH of the dilution buffer is from 5.5 to 6.5. In another embodiment, the pH of the dilution buffer is 6.0 ⁇ 0.2. In a specific embodiment, the pH of the dilution buffer is 6.0. In other embodiments, the dilution buffer has a pH selected from variations 6181 to 6403 found in Table 11.
  • buffering agent is present in the dilution buffer at a concentration of from 10 mM to 300 mM. In another embodiment, the concentration of buffering agent in the dilution buffer is between 10 mM to 200 mM. In other embodiments, the concentration of buffering agent in the dilution buffer is selected from variations 6404 to 6469 found in Table 12. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.
  • the furin dilution buffer includes calcium or another divalent metal cation.
  • the divalent cation is added as a salt, preferably a chloride salt.
  • the concentration of divalent cation (e.g., calcium) in the dilution buffer is from 0.1 mM to 10 mM, from 0.5 mM to 2 mM, or about 0.92 mM.
  • divalent cation from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1.5 mM to 7 mM, from 2 mM to 6 mM, from 2.5 mM to 5 mM, or from 3 mM to 4.5 mM divalent cation is used in the dilution buffer.
  • calcium salt it is preferably calcium chloride, but can also be other calcium salts such as calcium gluconate, calcium glubionate, or calcium gluceptate.
  • Divalent cations, including calcium can be included in combination with one or more other formulation components disclosed herein.
  • Non-limiting examples of divalent metal cations useful in the dilution buffers provided herein include calcium, barium, manganese, magnesium, cobalt, copper, nickel, and zinc.
  • the furin dilution buffer includes a sugar and/or sugar alcohol.
  • the dilution buffer contains a monosaccharide sugar.
  • the monosaccharide sugar is selected from the group consisting of a diose sugar, a triose sugar, a tetrose sugar, a pentose sugar, a hexose sugar, a heptose sugar, and an octose sugar.
  • the sugar is a pentose sugar, a hexose sugar, or a combination thereof.
  • the sugar is a hexose sugar.
  • the dilution buffer contains a disaccharide sugar.
  • the disaccharide sugar is selected from disaccharide sugars formed from pentose and/or hexose monosaccharides.
  • the sugar is selected from disaccharide sugars formed from hexose monosaccharides.
  • the sugar is sucrose, trehalose, or a combination thereof.
  • the sugar is sucrose.
  • the sugar is trehalose.
  • the sugar is formulated as trehalose dihydrate.
  • the dilution buffer contains a sugar alcohol.
  • the sugar alcohol is selected from glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol is mannitol.
  • the dilution buffer contains a mixture of sugar and sugar alcohol.
  • the mixture contains at least two of a monosaccharide, a disaccharide, and a sugar alcohol.
  • the mixture contains at least two of a pentose sugar, a hexose sugar, a disaccharide formed from pentose and/or hexose monosaccharides, and a sugar alcohol.
  • the mixture contains at least two of sucrose, trehalose, and mannitol.
  • the sugar or sugar alcohol is present in the dilution buffer at a concentration of from 2% to 20%, 2% to 17.5%, 2% to 15%, 2% to 12.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 5% to 20%, 5% to 17.5%, 5% to 15%, 5% to 12.5%, 5% to 10%, 7.5% to 20%, 7.5% to 17.5%, 7.5% to 15%, 7.5% to 12.5%, 10% to 20%, 10% to 17.5%, 10% to 15%, 4 ⁇ 2%, 5 ⁇ 2%, 6 ⁇ 2%, 7 ⁇ 2%, 8 ⁇ 2%, 9 ⁇ 2%, 10 ⁇ 2%, 11 ⁇ 2%, 12 ⁇ 2%, 13 ⁇ 2%, 14 ⁇ 2%, 15 ⁇ 2%, 16 ⁇ 2%, 17 ⁇ 2%, 18 ⁇ 2%, 3 ⁇ 1%, 4 ⁇ 1%, 5 ⁇ 1%, 6 ⁇ 1%, 7 ⁇ 1%, 8 ⁇ 1%, 9 ⁇ 1%, 10 ⁇ 1%, 11 ⁇ 1%, 12 ⁇ 1%, 13 ⁇ 1%, 14 ⁇ 1%, 15 ⁇ 1%, 16 ⁇ 1%, 17 ⁇ 1%, 18 ⁇ 1%, 3 ⁇ 1%
  • compositions of furin which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof.
  • a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol,
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the furin dilution buffer, or non-ionic surfactant solution added prior to dilution contains a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof.
  • a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the non-ionic surfactant is present in the dilution buffer at a concentration of from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm,
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the target concentration of non-ionic surfactant after dilution is from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 200 pp
  • the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester.
  • the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the inclusion of a non-ionic surfactant in a furin dilution buffer, or addition prior to dilution increases the furin activity recovered in a diluted furin composition by at least 10%, as compared to the activity recovered in a furin composition diluted without use of the non-ionic surfactant, or with the use of the non-ionic surfactant at a lower concentration.
  • the furin activity recovered after dilution using a non-ionic surfactant is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 2-fold, 3-fold, 4-fold, 5-fold, or more greater than the furin activity recovered after dilution without the non-ionic surfactant, or using a lower concentration of the non-ionic surfactant.
  • the highly stabilized furin (e.g., rfurin) formulations of the present disclosure show improved stability as compared to control formulations.
  • improved stability includes retention of a higher percentage of activity than control formulations in various stability assays.
  • Such assays can be used to determine if a formulation is a highly stabilized formulation.
  • the highly stabilized formulation has at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater activity than a control formulation when assessed by any of the stability assays discussed herein or known in the art.
  • furin (e.g., rfurin) formulations are tested under stressor conditions, such as storage at high temperature, agitation, freeze/thaw cycles, or some combination thereof. After such stressors, the formulations are assayed using any of the methods described herein or known in the art to determine the stability under these conditions.
  • furin activity assays are used to assess the stability of a formulation.
  • a furin activity assays involve a measurement of substrate cleavage.
  • the assay is performed, for example, on an ELISA plate.
  • the furin activity in a sample is determined from a standard curve created, for example, from a quality control (QC) furin standard.
  • the activity values are generally calculated as mean values from two dilutions, each in duplicate.
  • size exclusion chromatography is used to evaluate the stability of a furin (e.g., rfurin) formulation.
  • stability is indicated by the height of the peak corresponding to monomeric furin.
  • a reduction in monomer peak height means the product is being lost (unstable) and the generation of higher molecular weight peaks indicates that aggregation is occurring and lower molecular weight peaks means the product is degrading.
  • the relative peak height of a control furin formulation was 58.5%, but in a highly stabilized formulation, the relative peak height was 74.8% ( FIG. 48 ).
  • the highly stabilized formulation maintained the stability of furin in the composition and retained a larger percentage of the peak height as compared to that seen prior to storage at four days at a higher (37° C.) temperature.
  • FIG. 26 shows that after 5 days at 35° C., a greater percent of the signal is present as the intact furin molecule rather than the degraded species in the stabilized formulation (lane 11), as compared to a non-stabilized formulation (lane 5).
  • UV spectra are used to evaluate stability. Such assays are particularly useful when the stressor used is agitation.
  • the UV spectra can indicate the presence of aggregate—the presence of an aggregate is an indication of instability.
  • FIG. 36 shows a major displacement of the spectra upward (indicating aggregation) when the control formulation is agitated.
  • FIGS. 41 and 42 show the protective power of a highly stabilized formulation in that, with agitation, the spectra of the agitated sample is not displaced upward, but overlays the non-agitated sample, which indicates that aggregation was not formed when the formulation was placed under stress through agitation.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art.
  • Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.
  • Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols.
  • the stability of a control furin formulation was tested at three different temperatures: room temperature (e.g., 25° C.), 37° C., and 45° C. These studies at elevated temperatures were used to study accelerated stability, which is comparable to long term studies of stability at lower temperatures. The samples were tested at time zero, then after 1, 2, 3, 4, and 7 days of incubation. Analyses included the furin activity assay, SEC, and western blotting.
  • furin activity assay One method of assessing stability is through a furin activity assay.
  • the activity of furin is determined from the rate of a substrate cleavage.
  • the assay is performed, for example, on an ELISA plate.
  • the furin activity in a sample is determined from a standard curve created, for example, from a quality control rfurin standard. The activity values were calculated as mean values from two dilutions, each in duplicate.
  • rFurin stability at 37° C. in the highly stabilized formulation was to test rfurin stability in a stabilized formulation containing 75 ppm polysorbate 80. There was a small change in the composition of this sample because trehalose dihydrate (C 12 H 22 O 11 *2H 2 O, FW 378.33) was used, rather than, nonhydrated trehalose (C 12 H 22 O 11 , FW 342.30). Use of the trehalose dihydrate created a slightly more dilute sample. Agitation studies suggested that the polysorbate 80 concentration should be kept between 50 ppm and 100 ppm to provide the best protection against rfurin denaturation induced by agitation. In this study, a rfurin sample in test formulation #1 was spiked with polysorbate 80 to 75 ppm and incubated at 37° C. for four days. An rfurin sample formulated with the control formulation served as a control.
  • SEC Size exclusion chromatograph
  • test formulation #1 ( FIG. 48 ), containing trehalose, as compared to the control formulation, which does not contain a sugar or sugar alcohol.
  • FIG. 48 shows that test formulation #1 maintained 74.8% of rfurin monomer peak height of the starting solution after incubation at 37° C. for 4 days, as compared to the control formulation, which maintained only 58.5% of rfurin monomer peak height of the starting solution during the same incubation.
  • this data supports the ability to freeze and thaw the rFurin, not only once, but multiple times, with little to no loss of activity.
  • SEC analysis of the oligomeric state of furin in the control formulation showed no loss in monomeric content over the course of the freeze/thaw experiment ( FIG. 6 ).
  • rfurin in the control formulation is relatively stable for short periods at room temperature. Furthermore, loss of activity upon storage at elevated temperatures correlates to both rfurin degradation and aggregation. In addition, rfurin in the control formulation retained its activity well even after five freeze/thaw cycles.
  • Freeze/Thaw Study of rFurin in a highly stabilized Formulation The freeze/thaw study was carried out using the same set of samples as the previous Freeze/thaw study; the stability of rfurin in test formulations #2 (0 ppm polysorbate 80), #3 (10 ppm polysorbate 80), #4 (25 ppm polysorbate 80), #5 (50 ppm polysorbate 80), and #6 (100 ppm polysorbate 80) was evaluated.
  • Furin in the control formulation served as a control. Consistent with the prior analysis of the control formulation, analysis of furin activity ( FIG. 43 ) demonstrated no significant loss of activity after five freeze/thaw cycles in any of the tested formulations.
  • one potential mechanism of the agitation effect on rfurin samples is that it induces denaturation of rfurin, exposing hydrophobic surfaces.
  • the hydrophobic surfaces are attracted to other hydrophobic surfaces, such as other denatured rFurin proteins which induces aggregation, or causing adsorption to hydrophobic surfaces on sample tubes and materials.
  • agitation study was not designed to mimic the true process of rfurin use, but to overstress the sample.
  • a formulation of rfurin performs at least as well as the control formulation, and a highly stabilized formulation performs better than the control formulation.
  • rfurin in the control formulation is fairly resistant to agitation for a limited period of time. After two hours of vigorous agitation, such as might occur during a diafiltration step, more than 85% of rfurin was still active in the control formulation. However, after 17 hours of agitation, as might occur during shipping of a sample, less than 10% of the rfurin content remained active.
  • furin test formulations #2 to 6 which were created by spiking nine parts of a rFurin sample with one part of 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6 plus 10% w/w trehalose plus varied amounts of polysorbate 80.
  • the SEC profile for the agitated sample in the control formulation does not show any additional peaks compared to the non-agitated sample. This would suggest that surface adsorption was the main reason for the activity loss.
  • the UV absorption spectra show that the agitated sample has a slanted and elevated profile, which is a sign of light scattering and obscuration caused by the presence of aggregates. Light obscuration is generally caused by vary large aggregates that are more opaque than smaller molecules, which cause an elevation of the UV spectra. This phenomenon cannot be distinguished from absorption by a spectrophotometer because its detector measures the amount of light that passes through a sample. Since less light gets to the detector, it is reported as absorption; hence the absorption profile is elevated.
  • UV spectrophotometry analysis was conducted using the following equipment and materials:
  • the spectrophotometry analysis was conducted by first placing 0.5 mL of an appropriate formulation in a cuvette and measured as a blank. Then, 0.5 mL of a rfurin sample was placed in a cuvette, and the scan was taken from 240 nm to 400 nm.
  • rFurin samples were spiked with various buffers in order to adjust the pH to 5.0, 5.5, 6.0, 7.0 and 8.0.
  • sucrose and/or polysorbate 80 were added to the samples at pH 6.0, 7.0 and 8.0.
  • the samples were incubated at 37° C. and tested by the furin activity assay and SEC.
  • sucrose slowed down rfurin degradation and/or activity loss at 37° C. While sucrose is not used in downstream applications such as the rVWF process, other sugars and/or sugar alcohols are, such as mannitol. The addition of mannitol to the rfurin formulation, rather than sucrose, would therefore not introduce a new raw material to the vWF process. This study was conducted to determine whether the addition of mannitol would have a beneficial effect on rfurin stability. The test formulation also possessed an improved buffering capacity by addition of HEPES and acetic acid to final concentrations of 50 mM each at pH 6.0.
  • a stabilized formulation was created by spiking a rfurin sample with 10% of 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl 2 , pH 6, and then, adding either sucrose, mannitol, and/or polysorbate 80.
  • Analyses by both the furin activity assay ( FIG. 21 ) and SEC ( FIG. 22 ) showed that after four days of incubation at 37° C., mannitol and sucrose had similar stabilizing effects on rfurin formulations. Again, the effect of polysorbate 80 on these formulations was not significant.
  • the activity data show that the sample containing mannitol and polysorbate 80 after 4 days of incubation at 37° C. was better than other samples, not only does this sample not follow the overall pattern, but also the SEC analyses ( Figure) did not confirm it. It is likely that this result was an outlier.
  • a polyclonal antibody was used in this study instead of a monoclonal to increase detection of rFurin degradation products.
  • 5 ⁇ l of a sample was combined with 1 ⁇ l of 10 ⁇ DTT, and 4 ⁇ l of 4 ⁇ non-reducing dye.
  • the sample was heated at 70° C. to 75° C. for 10 minutes.
  • 10 ⁇ l of the sample was loaded to 4-20% SDS-PAGE gel, and run at 150 V for 60 to 70 minutes.
  • the protein gel was transferred to PVDF membrane using the iBLOT at 20 V for 6 minutes.
  • the PVDF membrane was then blocked with PBS buffer containing 0.1% TWEEN 20 and 3% BSA for 1 hour. The membrane was incubated for one hour in the primary antibody diluted 125 fold with the same buffer.
  • the membrane was washed with PBST buffer for five minutes, three times each.
  • the membrane was incubated for one hour in the secondary antibody diluted 10,000 fold with the same buffer as the primary antibody.
  • the incubation time was 1 hour.
  • the blot was washed using PBST buffer for five minutes, three times each.
  • the blot was developed in 10 mL of premixed chromagenic substrate (1 ml concentrate plus 9 ml of Stable Peroxidase Buffer) followed by the wash with water for 10 minutes.
  • the rFurin dilution study was carried out to compare a starting formulation and its diluent to a stabilized formulation and a stabilized diluent.
  • the study mimicked the dilution method that used in the rVWF maturation process. Briefly, rfurin BDS was passed through a filter followed by the addition of a 200-fold volume of the diluent.
  • the main difference between the test dilution method and the starting dilution method is that in the test (highly stabilized) method, 75 ppm polysorbate 80 is present in both the rfurin BDS and the diluent, while in the starting method, there is no polysorbate 80 in either the rfurin BDS or the diluent.
  • the test method recovered 80% of the expected rfurin activity, while the control method recovered only 27% (Table 19).
  • the rfurin sample was passed through a filter and followed by a 200-fold volume of a diluent. The filtrate was collected to a cylinder.
  • the combination of the stabilized rfurin formulation and the stabilized diluent increased rfurin recovery three to four times compared to the control method.
  • the improved recovery is, without being limited by theory, linked to the presence of polysorbate 80.
  • the stock buffer for the stabilized rfurin formulation (500 mM HEPES, 400 mM acetic acid, 1 mM CaCl 2 , pH 6.0) can be made consistently (Table 20).
  • the RSD's for pH and conductivity were ⁇ 0.3%.
  • the pH was slightly and indirectly correlated with temperature, dropping 0.014 units per degree Celsius, while conductivity was directly correlated with temperature, raising 0.40 mS/cm per degree C. ( FIG. 49 ).
  • the average temperature of the samples when the pH and conductivity were measured was 27.6° C. In manufacturing, the samples are read at 25 ⁇ 0.5° C. Based on the linear regressions in FIG. 48 , the expected pH and conductivity at 25° C. were calculated.
  • Reagent Buffer 1 Buffer 2 Buffer 3 mean sd rsd, % mean + 3 sd mean ⁇ 3 sd Water, L 0.85 0.85 0.85 HEPES, g 119.15 119.15 119.15 CaCl2, g 0.111 0.111 0.111 Acetic acid, mL 23 23 23 10M NaOH, mL 39.4 39.4 39.4 pH 6.011 6.003 6.005 6.006 0.004 0.07 6.019 5.994 Cond., mS/cm 21.23 21.15 21.11 21.16 0.06 0.29 21.35 20.98 Temp., C. 27.5 27.6 27.6 27.6 0.06 0.21 27.7 27.4

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006314A2 (fr) 1989-10-25 1991-05-16 H.B.T. Holland Biotechnology B.V. Preparation pharmaceutique a activite endoproteolytique; procede de traitement endoproteolytique des proteines (precurseurs) et de production (micro)biologique des proteines
WO1992009698A1 (fr) 1990-11-26 1992-06-11 Genetics Institute, Inc. Expression de pace dans des cellules hotes et procedes d'utilisation
US6210929B1 (en) 1995-11-24 2001-04-03 Baxter Aktiengesellschaft Fusion protein comprising a furin derivative or a derivative of a furin analogue and a heterologous sequence
US6596526B1 (en) 2000-06-09 2003-07-22 Baxter Aktiengesellschaft Furin polypeptides with improved characteristics
WO2005058283A2 (fr) 2003-12-19 2005-06-30 Novo Nordisk Health Care Ag Compositions stabilisees de polypeptides de facteur vii
EP1825865A2 (fr) 2006-02-24 2007-08-29 Dade Behring Marburg GmbH Préparations stabilisées de sérines endopeptidases, leur fabrication et utilisation
US20090181423A1 (en) * 2007-12-31 2009-07-16 Baxter International Inc. Substantially animal protein-free recombinant furin and methods for producing same
US20090304669A1 (en) 2007-05-22 2009-12-10 Baxter International Inc. Preparative purification process for human furin

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA965368B (en) * 1995-07-14 1997-01-14 Novo Nordisk As A pharmaceutical formulation
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US7033991B2 (en) * 2001-07-16 2006-04-25 Board Of Supervisors Of Louisiana State University And Agriculture And Mechanical College Inhibiting furin with polybasic peptides

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006314A2 (fr) 1989-10-25 1991-05-16 H.B.T. Holland Biotechnology B.V. Preparation pharmaceutique a activite endoproteolytique; procede de traitement endoproteolytique des proteines (precurseurs) et de production (micro)biologique des proteines
WO1992009698A1 (fr) 1990-11-26 1992-06-11 Genetics Institute, Inc. Expression de pace dans des cellules hotes et procedes d'utilisation
US6210929B1 (en) 1995-11-24 2001-04-03 Baxter Aktiengesellschaft Fusion protein comprising a furin derivative or a derivative of a furin analogue and a heterologous sequence
US6596526B1 (en) 2000-06-09 2003-07-22 Baxter Aktiengesellschaft Furin polypeptides with improved characteristics
WO2005058283A2 (fr) 2003-12-19 2005-06-30 Novo Nordisk Health Care Ag Compositions stabilisees de polypeptides de facteur vii
EP1825865A2 (fr) 2006-02-24 2007-08-29 Dade Behring Marburg GmbH Préparations stabilisées de sérines endopeptidases, leur fabrication et utilisation
US8124737B2 (en) * 2006-02-24 2012-02-28 Seimens Healthcare Diagnostics Products GmbH Stabilized preparations of serine endopeptidases, their preparation and use
US20090304669A1 (en) 2007-05-22 2009-12-10 Baxter International Inc. Preparative purification process for human furin
US20090181423A1 (en) * 2007-12-31 2009-07-16 Baxter International Inc. Substantially animal protein-free recombinant furin and methods for producing same

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Bravo, D.A. et al., "Accurate and Efficient Cleavage of the Human Insulin Proreceptor by the Human Proprotein-processing Protease Furin," The Journal of Biological Chemistry, Oct. 14, 1994, vol. 269, No. 41, pp. 25830-25837.
Hosaka, M. et al., "Arg-X-Lys/Arg-Arg Motif as a Signal for Precursor Cleavage Catalyzed by Furin within the Constitutive Secretory Pathway," The Journal of Biological Chemistry, Jul. 5, 1991, vol. 266, No. 19, pp. 12127-12130.
Human Recombinant Furin F2677. Datasheet [online]. Sigma Aldrich [retrieved on Feb. 26, 2013]. Retrieved from the Internet: <URL: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/7/f2677dat.Par.0001.File.tmp/f2677dat.pdf>. *
International Search Report for International Patent Application No. PCT/US2012/040790 mailed Nov. 5, 2012, 5 pages.
Molloy, S.S. et al., "Human Furin is a Calcium-dependent Serine Endoprotease That Recognizes the Sequence Arg-X-X-Arg and Efficiently Cleaves Anthrax Toxin Protective Antigen," The Journal of Biological Chemistry, Aug. 15, 1992, vol. 267, No. 23, pp. 16396-16402.

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